Layered evolution of gene expression in "superfast" muscles for courtship.

Identifying the molecular process of complex trait evolution is a core goal of biology. However, pinpointing the specific context and timing of trait-associated changes within the molecular evolutionary history of an organism remains an elusive goal. We study this topic by exploring the molecular basis of elaborate courtship evolution, which represents an extraordinary example of trait innovation. Within the behaviorally diverse radiation of Central and South American manakin birds, species from two separate lineages beat their wings together using specialized "superfast" muscles to generate a "snap" that helps attract mates. Here, we develop an empirical approach to analyze phylogenetic lineage-specific shifts in gene expression in the key snap-performing muscle and then integrate these findings with comparative transcriptomic sequence analysis. We find that rapid wing displays are associated with changes to a wide range of molecular processes that underlie extreme muscle performance, including changes to calcium trafficking, myocyte homeostasis and metabolism, and hormone action. We furthermore show that these changes occur gradually in a layered manner across the species history, wherein which ancestral genetic changes to many of these molecular systems are built upon by later species-specific shifts that likely finalized the process of display performance adaptation. Our study demonstrates the potential for combining phylogenetic modeling of tissue-specific gene expression shifts with phylogenetic analysis of lineage-specific sequence changes to reveal holistic evolutionary histories of complex traits.

The form, function, and evolutionary significance of neural aromatization.

Songbirds have emerged as exceptional research subjects for helping us appreciate and understand estrogen synthesis and function in brain. In the context of recognizing the vertebrate-wide importance of brain aromatase expression, in this review we highlight where we believe studies of songbirds have provided clarification and conceptual insight. We follow by focusing on more recent studies of aromatase and neuroestrogen function in the hippocampus and the pallial auditory processing region NCM of songbirds. With perspectives drawn from this body of work, we speculate that the evolution of enhanced neural estrogen signaling, including in the mediation of social behaviors, may have given songbirds the resilience to radiate into one of the most successful vertebrate groups on the planet.

Systems biology as a framework to understand the physiological and endocrine bases of behavior and its evolution-From concepts to a case study in birds.

Organismal behavior, with its tremendous complexity and diversity, is generated by numerous physiological systems acting in coordination. Understanding how these systems evolve to support differences in behavior within and among species is a longstanding goal in biology that has captured the imagination of researchers who work on a multitude of taxa, including humans. Of particular importance are the physiological determinants of behavioral evolution, which are sometimes overlooked because we lack a robust conceptual framework to study mechanisms underlying adaptation and diversification of behavior. Here, we discuss a framework for such an analysis that applies a "systems view" to our understanding of behavioral control. This approach involves linking separate models that consider behavior and physiology as their own networks into a singular vertically integrated behavioral control system. In doing so, hormones commonly stand out as the links, or edges, among nodes within this system. To ground our discussion, we focus on studies of manakins (Pipridae), a family of Neotropical birds. These species have numerous physiological and endocrine specializations that support their elaborate reproductive displays. As a result, manakins provide a useful example to help imagine and visualize the way systems concepts can inform our appreciation of behavioral evolution. In particular, manakins help clarify how connectedness among physiological systems-which is maintained through endocrine signaling-potentiate and/or constrain the evolution of complex behavior to yield behavioral differences across taxa. Ultimately, we hope this review will continue to stimulate thought, discussion, and the emergence of research focused on integrated phenotypes in behavioral ecology and endocrinology.

11ß hydroxysteroid dehydrogenases regulate circulating glucocorticoids but not central gene expression.

Regulation of glucocorticoids (GCs), important mediators of physiology and behavior at rest and during stress, is multi-faceted and dynamic. The 11ß hydroxysteroid dehydrogenases 11ß-HSD1 and 11ß-HSD2 catalyze the regeneration and inactivation of GCs, respectively, and provide peripheral and central control over GC actions in mammals. While these enzymes have only recently been investigated in just two songbird species, central expression patterns suggest that they may function differently in birds and mammals, and little is known about how peripheral expression regulates circulating GCs. In this study, we utilized the 11ß-HSD inhibitor carbenoxolone (CBX) to probe the functional effects of 11ß-HSD activity on circulating GCs and central GC-dependent gene expression in the adult zebra finch (Taeniopygia guttata). Peripheral CBX injection produced a marked increase in baseline GCs 60 min after injection, suggestive of a dominant role for 11ß-HSD2 in regulating circulating GCs. In the adult zebra finch brain, where 11ß-HSD2 but not 11ß-HSD1 is expressed, co-incubation of micro-dissected brain regions with CBX and stress-level GCs had no impact on expression of several GC-dependent genes. These results suggest that peripheral 11ß-HSD2 attenuates circulating GCs, whereas central 11ß-HSD2 has little impact on gene expression. Instead, rapid 11ß-HSD2-based regulation of local GC levels might fine-tune membrane GC actions in brain. These results provide new insights into the dynamics of GC secretion and action in this important model organism.

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

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

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

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

Evolution of the androgen-induced male phenotype.

The masculine reproductive phenotype varies significantly across vertebrates. As a result, biologists have long recognized that many of the mechanisms that support these phenotypes-particularly the androgenic system-is evolutionarily labile, and thus susceptible to the effects of selection for different traits. However, exactly how androgenic signaling systems vary in a way which results in dramatically different functional outputs, remain largely unclear. We explore this topic here by outlining four key-but non-mutually exclusive-hypotheses that propose how the mechanisms of androgenic signaling might change over time to potentiate the emergence of phenotypical variation in masculine behavior and physiology. We anchor this framework in a review of our own studies of a tropical bird called the golden-collared manakin (Manacus vitellinus), which has evolved an exaggerated acrobatic courtship display that is heavily androgen-dependent. The result is an example of how the cellular basis of androgenic action can be modified to support a unique reproductive repertoire. We end this review by highlighting a broad pathway forward to further pursue the intricate ways by which the mechanisms of hormone action evolve to support processes of adaptation and animal design.

Muscle, a conduit to brain for hormonal control of behavior.

SBN Elsevier Lecture Investigation into mechanisms whereby hormones control behavior often starts with actions on central nervous system (CNS) motivation and motor systems and is followed by assessment of CNS drive of coordinated striated muscle contractions. Here we turn this perspective on its head by discussing ways in which hormones might first act on muscle that then secondarily drive upstream the evolution and function of the CNS. While there is a lengthy history for consideration of this perspective, newly discovered properties of muscle signaling reveal novel mechanisms that may well be captured by endocrine systems and thus of interest to behavioral endocrinologists.

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

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

Neuromuscular mechanisms of an elaborate wing display in the golden-collared manakin (Manacus vitellinus).

Many species perform elaborate physical displays to court mates and compete with rivals, but the biomechanical mechanisms underlying such behavior are poorly understood. We address this issue by studying the neuromuscular origins of display behavior in a small tropical passerine bird, the golden-collared manakin (Manacus vitellinus). Males of this species court females by dancing around the forest floor and rapidly snapping their wings together above their back. Using radio-telemetry, we collected electromyographic (EMG) recordings from the three main muscles that control avian forelimb movement, and found how these different muscles are activated to generate various aspects of display behavior. The muscle that raises the wing (supracoracoideus, SC) and the primary muscle that retracts the wing (scapulohumeralis caudalis, SH) were activated during the wing-snap, whereas the pectoralis (PEC), the main wing depressor, was not. SC activation began before wing elevation commenced, with further activation occurring gradually. By contrast, SH activation was swift, starting soon after wing elevation and peaking shortly after the snap. The intensity of this SH activation was comparable to that which occurs during flapping, whereas the SC activation was much lower. Thus, light activation of the SC likely helps position the wings above the back, so that quick, robust SH activation can drive these appendages together to generate the firecracker-like snap sonation. This is one of the first looks at the neuromuscular mechanisms that underlie the actuation of a dynamic courtship display, and it demonstrates that even complex, whole-body display movements can be studied with transmitter-aided EMG techniques.

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

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

Expression of 5α- and 5ß-reductase in spinal cord and muscle of birds with different courtship repertoires.

BACKGROUND: Through the actions of one or more isoforms of the enzyme 5α-reductase in many male reproductive tissues, circulating testosterone (T) undergoes metabolic conversion into 5α-dihydrotestosterone (DHT), which binds to and activates androgen receptors (AR) with greater potency than T. In birds, T is also subject to local inactivation into 5ß-DHT by the enzyme 5ß-reductase. Male golden-collared manakins perform an androgen-dependent and physically elaborate courtship display, and these birds express androgen receptors in skeletal muscles and spinal cord at levels far greater than those expressed in species with more limited courtship routines, including male zebra finches. To determine if local T metabolism facilitates or impedes activation of male manakin courtship, we examined expression of two isoforms of 5α-reductase, as well as 5ß-reductase, in forelimb muscles and spinal cords of males and females of the two aforementioned species. RESULTS: We found that all enzymes were expressed in all tissues, with patterns that partially predict a functional role for 5α-reductase in these birds, especially in both muscle and spinal cord of male manakins. Moreover, we found that 5ß-reductase was markedly different between species, with far lower levels in golden-collared manakins, compared to zebra finches. Thus, modification to neuromuscular deactivation of T may also play a functional role in adaptive behavioral modulation. CONCLUSIONS: Given that such a role for 5α-reductase in androgen-sensitive mammalian skeletal muscle is in dispute, our data suggest that, in birds, local metabolism may play a key role in providing active androgenic substrates to peripheral neuromuscular systems. Similarly, we provide the first evidence that 5ß-reductase is expressed broadly through an organism and may be an important factor that regulates androgenic modulation of neuromuscular functioning.

Determination of the wingsnap sonation mechanism of the golden-collared manakin (Manacus vitellinus).

Male golden-collared manakins (Manacus vitellinus), small suboscine passeriform birds of Panamanian forests, communicate acoustically using a variety of non-vocal sonations. The most prominent sonations are single or multiple intense 'wingsnaps' with a dominant acoustic frequency around 5 kHz. Several hypotheses have been proposed addressing the source of the sound, ranging from purely aerodynamic origins (due to a rapid jet of air formed by the wings or by a 'whiplike' motion) to purely structural origins (such as physical contact of the wings), but without definitive assessment. Using anatomical analysis as well as high-speed video and synchronized audio recordings, we show that compared with related species, M. vitellinus radii are morphologically unique and confirm that they collide over the back of the bird at the moment (±1 ms) the wingsnap is produced. Using aeroacoustic theory, we quantitatively estimate the acoustic signatures from several sonation mechanisms. We conclude that only the physical contact hypothesis, wherein the wing collisions create the sound, is consistent with the measured sonation.

Determinants and significance of corticosterone regulation in the songbird brain.

Songbirds exhibit significant adult neuroplasticity that, together with other neural specializations, makes them an important model system for neurobiological studies. A large body of work also points to the songbird brain as a significant target of steroid hormones, including corticosterone (CORT), the primary avian glucocorticoid. Whereas CORT positively signals the brain for many functions, excess CORT may interfere with natural neuroplasticity. Consequently, mechanisms may exist to locally regulate CORT levels in brain to ensure optimal concentrations. However, most studies in songbirds measure plasma CORT as a proxy for levels at target tissues. In this paper, we review literature concerning circulating CORT and its effects on behavior in songbirds, and discuss recent work suggesting that brain CORT levels are regulated independently of changes in adrenal secretion. We review possible mechanisms for CORT regulation in the avian brain, including corticosteroid-binding globulins, p-glycoprotein activity in the blood-brain barrier and CORT metabolism by the 11ß hydroxysteroid dehydrogenases. Data supporting a role for CORT regulation within the songbird brain have only recently begun to emerge, suggesting that this is an avenue for important future research.

Sex, estradiol, and spatial memory in a food-caching corvid.

Estrogens significantly impact spatial memory function in mammalian species. Songbirds express the estrogen synthetic enzyme aromatase at relatively high levels in the hippocampus and there is evidence from zebra finches that estrogens facilitate performance on spatial learning and/or memory tasks. It is unknown, however, whether estrogens influence hippocampal function in songbirds that naturally exhibit memory-intensive behaviors, such as cache recovery observed in many corvid species. To address this question, we examined the impact of estradiol on spatial memory in non-breeding Western scrub-jays, a species that routinely participates in food caching and retrieval in nature and in captivity. We also asked if there were sex differences in performance or responses to estradiol. Utilizing a combination of an aromatase inhibitor, fadrozole, with estradiol implants, we found that while overall cache recovery rates were unaffected by estradiol, several other indices of spatial memory, including searching efficiency and efficiency to retrieve the first item, were impaired in the presence of estradiol. In addition, males and females differed in some performance measures, although these differences appeared to be a consequence of the nature of the task as neither sex consistently out-performed the other. Overall, our data suggest that a sustained estradiol elevation in a food-caching bird impairs some, but not all, aspects of spatial memory on an innate behavioral task, at times in a sex-specific manner.

Hormones and the neuromuscular control of courtship in the golden-collared manakin (Manacus vitellinus).

Many animals engage in spectacular courtship displays, likely recruiting specialized neural, hormonal and muscular systems to facilitate these performances. Male golden-collared manakins (Manacus vitellinus) of Panamanian rainforests perform physically elaborate courtship displays that include novel forms of visual and acoustic signaling. We study the behavioral neuroendocrinology of this male's courtship, combining field behavioral observations with anatomical, biochemical and molecular laboratory-based studies. Seasonally, male courtship is activated by testosterone with little correspondence between testosterone levels and display intensity. Females prefer males whose displays are exceptionally frequent, fast and accurate. The activation of androgen receptors (AR) is crucial for optimal display performance, with AR expressed at elevated levels in several neuromuscular tissues. Apparently, courtship enlists an elaborate androgen-dependent network that includes spinal motoneurons, skeletal muscles and somatosensory systems. This work highlights the value of studying non-traditional species to illuminate physiological adaptations and, hopefully, stimulates future research on other species with complex behaviors.

Establishing regional specificity of neuroestrogen action.

The specificity of estrogen signaling in brain is defined at one level by the types and distributions of receptor molecules that are activated by estrogens. At another level, as our understanding of the neurobiology of the estrogen synthetic enzyme aromatase has grown, questions have emerged as to how neuroactive estrogens reach specific target receptors in functionally relevant concentrations. Here we explore the spatial specificity of neuroestrogen signaling with a focus on studies of songbirds to provide perspective on some as-yet unresolved questions. Studies conducted in both male and female songbirds have helped to clarify these interesting facets of neuroestrogen physiology.

Peripheral androgen action helps modulate vocal production in a suboscine passerine.

Androgenic activation of intracellular androgen receptors (AR) influences avian vocal production, though this has largely been investigated at the level of the brain. We investigated the influence of predominantly peripheral AR on vocal output in wild Golden-collared Manakins (Manacus vitellinus). In this suboscine species, males court females by performing acrobatic displays and by producing relatively simple chee-poo vocalizations. To assess whether peripheral AR influences the acoustic structure of these vocal signals, we treated reproductively active adult males with the peripherally selective antiandrogen bicalutamide and then measured phonation performance. Inhibiting AR outside of the central nervous system increased the duration of the chee note and decreased the fundamental frequency of the poo note. This treatment caused no discernable change to chee-poo frequency modulation or entropy. Our results show that activation of peripheral AR mediates note-specific changes to temporal and pitch characteristics of the Golden-collared Manakin's main sexual call. Thus, our study provides one of the first demonstrations that androgenic action originating outside of the brain and likely on musculoskeletal targets can modulate avian vocal production.