Expression of novel androgen receptors in three GnRH neuron subtypes in the cichlid brain.

Within a social hierarchy, an individuals' social status determines its physiology and behavior. In A. burtoni, subordinate males can rise in rank to become dominant, which is accompanied by the upregulation of the entire HPG axis, including activation of GnRH1 neurons, a rise in circulating androgen levels and the display of specific aggressive and reproductive behaviors. Cichlids possess two other GnRH subtypes, GnRH2 and GnRH3, the latter being implicated in the display of male specific behaviors. Interestingly, some studies showed that these GnRH neurons are responsive to fluctuations in circulating androgen levels, suggesting a link between GnRH neurons and androgen receptors (ARs). Due to a teleost-specific whole genome duplication, A. burtoni possess two AR paralogs (ARα and ARß) that are encoded by two different genes, ar1 and ar2, respectively. Even though social status has been strongly linked to androgens, whether ARα and/or ARß are present in GnRH neurons remains unclear. Here, we used immunohistochemistry and in situ hybridization chain reaction (HCR) to investigate ar1 and ar2 expression specifically in GnRH neurons. We find that all GnRH1 neurons intensely express ar1 but only a few of them express ar2, suggesting the presence of genetically-distinct GnRH1 subtypes. Very few ar1 and ar2 transcripts were found in GnRH2 neurons. GnRH3 neurons were found to express both ar genes. The presence of distinct ar genes within GnRH neuron subtypes, most clearly observed for GnRH1 neurons, suggests differential control of these neurons by androgenic signaling. These findings provide valuable insight for future studies aimed at disentangling the androgenic control of GnRH neuron plasticity and reproductive plasticity across teleosts.

Androgen receptor deficiency is associated with reduced aromatase expression in the ventromedial hypothalamus of male cichlids.

Social behaviors are regulated by sex steroid hormones, such as androgens and estrogens. However, the specific molecular and neural processes modulated by steroid hormones to generate social behaviors remain to be elucidated. We investigated whether some actions of androgen signaling in the control of social behavior may occur through the regulation of estradiol synthesis in the highly social cichlid fish, Astatotilapia burtoni. Specifically, we examined the expression of cyp19a1, a brain-specific aromatase, in the brains of male A. burtoni lacking a functional ARα gene (ar1), which was recently found to be necessary for aggression in this species. We found that cyp19a1 expression is higher in wild-type males compared to ar1 mutant males in the anterior tuberal nucleus (ATn), the putative fish homolog of the mammalian ventromedial hypothalamus, a brain region that is critical for aggression across taxa. Using in situ hybridization chain reaction, we determined that cyp19a1+ cells coexpress ar1 throughout the brain, including in the ATn. We speculate that ARα may modulate cyp19a1 expression in the ATn to govern aggression in A. burtoni. These studies provide novel insights into the hormonal mechanisms of social behavior in teleosts and lay a foundation for future functional studies.

Sex differences in aggression and its neural substrate in a cichlid fish.

Aggression is ubiquitous among social species and functions to maintains social dominance hierarchies. The African cichlid fish Astatotilapia burtoni is an ideal study species for studying aggression due to their unique and flexible dominance hierarchy. However, female aggression in this species and the neural mechanisms of aggression in both sexes is not well understood. To further understand the potential sex differences in aggression in this species, we characterized aggression in male and female A. burtoni in a mirror assay. We then quantified neural activation patterns in brain regions of the social behavior network (SBN) to investigate if differences in behavior are reflected in the brain with immunohistochemistry by detecting the phosphorylated ribosome marker phospho-S6 ribosomal protein (pS6), a marker for neural activation. We found that A. burtoni perform both identical and sex-specific aggressive behaviors in response to a mirror assay. We observed sex differences in pS6 immunoreactivity in the Vv, a homolog of the lateral septum in mammals. Males but not females had higher ps6 immunoreactivity in the ATn after the aggression assay. The ATn is a homolog of the ventromedial hypothalamus in mammals, which is strongly implicated in the regulation of aggression in males. Several regions also have higher pS6 immunoreactivity in negative controls than fish exposed to a mirror, implicating a role for inhibitory neurons in suppressing aggression until a relevant stimulus is present. Male and female A. burtoni display both similar and sexually dimorphic behavioral patterns in aggression in response to a mirror assay. There are also sex differences in the corresponding neural activation patterns in the SBN. In mirror males but not females, the ATn clusters with the POA, revealing a functional connectivity of these regions that is triggered in an aggressive context in males. These findings suggest that distinct neural circuitry underlie aggressive behavior in male and female A. burtoni, serving as a foundation for future work investigating the molecular and neural underpinnings of sexually dimorphic behaviors in this species to reveal fundamental insights into understanding aggression.

A phylogenetics-based nomenclature system for steroid receptors in teleost fishes.

Teleost fishes have emerged as tractable models for studying the neuroendocrine regulation of social behavior via molecular genetic techniques, such as CRISPR/Cas9 gene editing. Moreover, teleosts provide an opportunity to investigate the evolution of steroid receptors and their functions, as species within this lineage possess novel steroid receptor paralogs that resulted from a teleost-specific whole genome duplication. Although teleost fishes have grown in popularity as models for behavioral neuroendocrinology, there is not a consistent nomenclature system for steroid receptors and their genes, which may impede a clear understanding of steroid receptor paralogs and their functions. Here, we used a phylogenetic approach to assess the relatedness of protein sequences encoding steroid receptor paralogs in 18 species from 12 different orders of the Infraclass Teleostei. While most similarly named sequences grouped based on the established phylogeny of the teleost lineage, our analysis revealed several inconsistencies in the nomenclature of steroid receptor paralogs, particularly for sequences encoding estrogen receptor beta (ERß). Based on our results, we propose a nomenclature system for teleosts in which Greek symbols refer to proteins and numbers refer to genes encoding different subtypes of steroid receptors within the five major groups of this nuclear receptor subfamily. Collectively, our results bridge a critical gap by providing a cohesive naming system for steroid receptors in teleost fishes, which will serve to improve communication, promote collaboration, and enhance our understanding of the evolution and function of steroid receptors across vertebrates.

Genetic dissection of steroid-hormone modulated social behavior: Novel paralogous genes are a boon for discovery.

Research across species has led to important discoveries on the functions of steroid hormones in the regulation of behavior. However, like in many fields, advancements in transgenic and mutagenic technology allowed mice to become the premier genetic model for conducting many experiments to understand how steroids control social behavior. Since there has been a general lack of parallel methodological developments in other species, many of the findings cannot be generalized. This is especially the case for teleost fish, in which a whole-genome duplication produced novel paralogs for key steroid hormone signaling genes. In this review, we summarize technical advancements over the history of the field of neuroendocrinology that have led to important insights in our understanding of the control of social behavior by steroids. We demonstrate that early mouse genetic models to understand these mechanisms suffered from several issues that were remedied by more precise transgenic technological advancements. We then highlight the importance of CRISPR/Cas9 gene editing tools that will in time bridge the gap between mice and non-traditional model species for understanding principles of steroid hormone action in the modulation of social behavior. We specifically highlight the role of teleost fish in bridging this gap because they are 1) highly genetically tractable and 2) provide a novel advantage in achieving precise genetic control. The field of neuroendocrinology is entering a new "gene editing revolution" that will lead to novel discoveries about the roles of steroid hormones in the regulation and evolutionary trajectories of social behavior.

Social regulation of immediate early gene induction in gonadotropin releasing-hormone 1 neurons and singing behavior in canaries (Serinus canaria).

Social cues modulate the neuroendocrine control of reproduction. However, the neural systems involved in the integration of social cues are not well described. Gonadotropin-releasing hormone 1 (GnRH1) cells in the preoptic area (POA) are the final common node that links the brain with peripheral reproductive physiology. These experiments investigated whether induction of the immediate early gene, EGR1, in anatomically localized GnRH1 cell populations in Border canaries is regulated by the social environment. First, we characterized behavioral modifications in singing behavior and found males paired with a female for 2 weeks significantly reduced many aspects of singing behavior. However, paired males had a significantly higher percentage of GnRH1 cells co-labeled with EGR1. The second experiment manipulated the social environment by pairing males and females in mixed sex dyads, same sex dyads or housed birds in isolation. Only when birds are paired in mixed sex dyads was there a significantly greater percentage of GnRH1 cells expressing EGR1 cells. Increased GnRH1-EGR1 co-expression was localized to the rostral POA. These data reveal that discrete GnRH1 cells are involved in the neural integration of specific social cues and support the hypothesis that the POA exhibits functional topography related to courtship and sexual behaviors.

Control of testes mass by androgen receptor paralogs in a cichlid.

Steroid hormones play numerous important and diverse roles in the differentiation and development of vertebrate primary and secondary reproductive characteristics. However, the exact role of androgen receptors-which bind circulating androgens-in this regulatory pathway is unclear. Teleost fishes further complicate this question by having two paralogs of the androgen receptor (ARα and ARß) resulting from a duplication of their ancestral genome. We investigated the functional role of these two ARs on adult testes mass, by eliminating receptor function of one or both paralogs using CRISPR/Cas9 genome edited Astatotilapia burtoni, an African cichlid fish. Fish with two or more functional AR alleles were more likely to be male compared to fish with one or fewer, suggesting that the two paralogs may play redundant roles in the A. burtoni sex determination system. We replicated previous work showing that fish lacking functional ARß possess testes smaller than wild-type fish, while fish lacking ARα possess testes larger than wild-type fish. However, we found novel evidence supporting a complex relationship between the two AR paralogs in the regulation of testes mass. For instance, the effects of ARα mutation on testes mass are eliminated in homozygous ARß mutants but the reverse is not true. These results suggest a dynamic relationship between these two AR paralogs where ARß functions may be permissive to ARα functions in the control of testes mass. This mechanism may contribute to the robust physiological plasticity displayed by A. burtoni and other social teleost fishes.

Modular genetic control of social status in a cichlid fish.

Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fish Astatotilapia burtoni is a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. Dominant A. burtoni males possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status in A. burtoni is in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication, A. burtoni possess two androgen receptor (AR) paralogs, ARα and ARß, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutant A. burtoni and performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find that ARß, but not ARα, is required for testes growth and bright coloration, while ARα, but not ARß, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status in A. burtoni is inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity in A. burtoni and other species whose social status relies on social experience.

How does testosterone act to regulate a multifaceted adaptive response? Lessons from studies of the avian song system.

In male songbirds, song functions to attract a mate or to defend a territory; it is therefore often produced in the context of reproduction. Testosterone of gonadal origin increases during the reproductive phase of the annual cycle and significantly enhances song production, as well as song development, via effects on song crystallisation. The neural control of birdsong production and learning is highly modular. We implanted testosterone or androgen antagonists into specific brain regions or in the periphery of castrated male canaries and, in this way, identified how androgen signalling in specific locations regulates a variety of birdsong features. For example, castrated male canaries treated with testosterone in the preoptic area only and exposed to long days sing at high rates compared to castrated male canaries not treated with testosterone. However, these birds with testosterone in the preoptic area still produce songs with substantially lower song stereotypy and amplitude; these features are controlled by testosterone acting in the song control nuclei HVC and robust nucleus of the arcopallium. Specific aspects of the learned singing behaviour are thus regulated by androgens acting at multiple levels in the brain in a non-redundant fashion. The action of testosterone in the preoptic area is related to the hormonal regulation of the motivation to sing but not to various aspects of song performance. Multiple aspects of song quality are instead precisely regulated by steroids acting in distinct song control nuclei. Females exert a strong choice for specific features of male song in canaries and this choice is influenced by the endocrine state of the female. The female song system is also involved in song production, as well as song perception, although the specificity of this hormone action has not yet been investigated.

Hormonal regulation of social ascent and temporal patterns of behavior in an African cichlid.

For many species, social rank determines which individuals perform certain social behaviors and when. Higher ranking or dominant (DOM) individuals maintain status through aggressive interactions and perform courtship behaviors while non-dominant (ND) individuals do not. In some species ND individuals ascend (ASC) in social rank when the opportunity arises. Many important questions related to the mechanistic basis of social ascent remain to be answered. We probed whether androgen signaling regulates social ascent in male Astatotilapia burtoni, an African cichlid whose social hierarchy can be readily controlled in the laboratory. As expected, androgen receptor (AR) antagonism abolished reproductive behavior during social ascent. However, we discovered multiple AR- and status-dependent temporal behavioral patterns that typify social ascent and dominance. AR antagonism in ASC males increased the time between successive behaviors compared to DOM males. Socially ascending males, independent of AR activation, were more likely than DOM males to follow aggressive displays with another aggressive display. Further analyses revealed differences in the sequencing of aggressive and courtship behaviors, wherein DOM males were more likely than ASC males to follow male-directed aggression with courtship displays. Strikingly, this difference was driven mostly by ASC males taking longer to transition from aggression to courtship, suggesting ASC males can perform certain DOM-typical temporal behavioral patterns. Our results indicate androgen signaling is necessary for social ascent and hormonal signaling and social experience may shape the full suite of DOM-typical behavioral patterns.

The regulation of birdsong by testosterone: Multiple time-scales and multiple sites of action.

Contribution to Special Issue on Fast effects of steroids. Sex steroid hormones act during early development to shape the circuitry upon which these same hormones act in adulthood to control behavioral responses to various stimuli. The "organizational" vs. "activational" distinction was proposed to explain this temporal difference in hormone action. In both of these cases steroids were thought to act genomically over a time-scale of days to weeks. However, sex steroids can affect behavior over short (e.g., seconds or minutes) time-scales. Here, we discuss how testosterone controls birdsong via actions at different sites and over different time-scales, with an emphasis on this process in canaries (Serinus canaria). Our work shows that testosterone in the medial preoptic nucleus regulates the motivation to sing, but not aspects of song performance. Instead, different aspects of song performance are regulated by long-term actions of testosterone in steroid-sensitive cortical-like brain regions and the syrinx, the avian vocal production organ. On the other hand, acute aromatase inhibition rapidly reduces the availability of estrogens and this reduction is correlated with reductions in the motivation to sing and song performance. Thus, testosterone and its estrogenic metabolites regulate distinct features of birdsong depending on the site and temporal window of action. The number of brain areas expressing androgen receptors is higher in species producing learned vocalization as compared to species that produce unlearned calls. An appealing scenario is that rapid effects of steroids in specific brain regions is a derived trait secondary to the widespread genomic effects of steroids in systems where steroids coordinate morphological, physiological, and behavioral traits.

Sex differences in hippocampal mineralocorticoid and glucocorticoid receptor mRNA expression in response to acute mate pair separation in zebra finches (Taeniopygia guttata).

Mate separation has been shown to mediate changes in physiological and behavioral processes via activation of the hypothalamo-pituitary-adrenal (HPA) axis in both mammalian and avian species. To elucidate the neural mechanisms associated with changes in the HPA axis in response to social stress, we investigated the effects of mate pair separation on circulating corticosterone concentrations as well as gene expression levels of mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and corticotropin releasing hormone (CRH) in the hypothalamus and hippocampus of both male and female zebra finches, a species that forms strong pair bonds. Zebra finches (Taeniopygia guttata) were housed three to a cage (a mated pair plus a stimulus female), and were assigned to one of three new housing treatment groups: (1) male or female removed from their respective mate and placed in a cage with a new opposite sex conspecific and stimulus female (2) male or female that remained with their mate, but a new stimulus female was introduced, or (3) the subjects were handled but not separated from their mate or the stimulus female. After 48 hr in the new housing condition, we observed significant increases in plasma corticosterone concentrations in response to both mate pair and stimulus female separation. No significant differences in MR, GR, or CRH mRNA expression in the hypothalamus were observed in response to any treatment for both males and females. Females exhibited a significant up regulation in hippocampal MR, but not GR mRNA, whereas males exhibited a significant down regulation of both hippocampal MR and GR mRNA in response to mate pair separation. Thus, the hippocampus appears to play a key role in regulating sex specific responses to social stressors.

The Value of Comparative Animal Research: Krogh's Principle Facilitates Scientific Discoveries.

Biomedical research is dominated by relatively few nonhuman animals to investigate healthy and disease conditions. Research has overrelied on these models due to their well-described genomes, the capability to control specific genes, and the high rate of reproduction. However, recent advances in large-scale molecular sequencing experiments have revealed, in some cases, the limited similarities in experimental outcomes observed in common rodents (i.e., mice) compared with humans. The value of more varied comparative animal models includes examples such as long-term body weight regulation in seasonally breeding hamsters as a means to help understand the obesity epidemic, vocal learning in songbirds to illuminate language acquisition and maintenance, and reproduction in cichlid fish to discover novel genes conserved in humans. Studying brain genes in prairie voles and cichlids advanced knowledge about social behavior. Taken together, experiments on diverse animal species highlight nontraditional systems for advancing our understanding of human health and well-being.

Dissociable Effects on Birdsong of Androgen Signaling in Cortex-Like Brain Regions of Canaries.

The neural basis of how learned vocalizations change during development and in adulthood represents a major challenge facing cognitive neuroscience. This plasticity in the degree to which learned vocalizations can change in both humans and songbirds is linked to the actions of sex steroid hormones during ontogeny but also in adulthood in the context of seasonal changes in birdsong. We investigated the role of steroid hormone signaling in the brain on distinct features of birdsong using adult male canaries (Serinus canaria), which show extensive seasonal vocal plasticity as adults. Specifically, we bilaterally implanted the potent androgen receptor antagonist flutamide in two key brain regions that control birdsong. We show that androgen signaling in the motor cortical-like brain region, the robust nucleus of the arcopallium (RA), controls syllable and trill bandwidth stereotypy, while not significantly affecting higher order features of song such syllable-type usage (i.e., how many times each syllable type is used) or syllable sequences. In contrast, androgen signaling in the premotor cortical-like brain region, HVC (proper name), controls song variability by increasing the variability of syllable-type usage and syllable sequences, while having no effect on syllable or trill bandwidth stereotypy. Other aspects of song, such as the duration of trills and the number of syllables per song, were also differentially affected by androgen signaling in HVC versus RA. These results implicate androgens in regulating distinct features of complex motor output in a precise and nonredundant manner.SIGNIFICANCE STATEMENT Vocal plasticity is linked to the actions of sex steroid hormones, but the precise mechanisms are unclear. We investigated this question in adult male canaries (Serinus canaria), which show extensive vocal plasticity throughout their life. We show that androgens in two cortex-like vocal control brain regions regulate distinct aspects of vocal plasticity. For example, in HVC (proper name), androgens regulate variability in syntax but not phonology, whereas androgens in the robust nucleus of the arcopallium (RA) regulate variability in phonology but not syntax. Temporal aspects of song were also differentially affected by androgen signaling in HVC versus RA. Thus, androgen signaling may reduce vocal plasticity by acting in a nonredundant and precise manner in the brain.

Aromatase inhibition rapidly affects in a reversible manner distinct features of birdsong.

Recent evidence has implicated steroid hormones, specifically estrogens, in the rapid modulation of cognitive processes. Songbirds have been a useful model system in the study of complex cognitive processes including birdsong, a naturally learned vocal behavior regulated by a discrete steroid-sensitive telencephalic circuitry. Singing behavior is known to be regulated by long-term actions of estrogens but rapid steroid modulation of this behavior has never been examined. We investigated if acute actions of estrogens regulate birdsong in canaries (Serinus canaria). In the morning, male canaries sing within minutes after light onset. Birds were injected with fadrozole, a potent aromatase inhibitor, or vehicle within 2-5 minutes after lights on to implement a within-subjects experimental design. This single injection of fadrozole reduced the motivation to sing as well as song acoustic stereotypy, a measure of consistency over song renditions, on the same day. By the next day, however, all song measures that were affected had returned to baseline. This study indicates that estrogens also act in a rapid fashion to regulate two distinct features of song, a learned vocal behavior.

Pleiotropic Control by Testosterone of a Learned Vocal Behavior and Its Underlying Neuroplasticity(1,2,3).

Steroid hormones coordinate multiple aspects of behavior and physiology. The same hormone often regulates different aspects of a single behavior and its underlying neuroplasticity. This pleiotropic regulation of behavior and physiology is not well understood. Here, we investigated the orchestration by testosterone (T) of birdsong and its neural substrate, the song control system. Male canaries were castrated and received stereotaxic implants filled with T in select brain areas. Implanting T solely in the medial preoptic nucleus (POM) increased the motivation to sing, but did not enhance aspects of song quality such as acoustic structure and stereotypy. In birds implanted with T solely in HVC (proper name), a key sensorimotor region of the song control system, little or no song was observed, similar to castrates that received no T implants of any sort. However, implanting T in HVC and POM simultaneously rescued all measures of song quality. Song amplitude, though, was still lower than what was observed in birds receiving peripheral T treatment. T in POM enhanced HVC volume bilaterally, likely due to activity-dependent changes resulting from an enhanced song rate. T directly in HVC, without increasing song rate, enhanced HVC volume on the ipsilateral side only. T in HVC enhanced the incorporation and recruitment of new neurons into this nucleus, while singing activity can independently influence the incorporation of new neurons into HVC. These results have broad implications for how steroid hormones integrate across different brain regions to coordinate complex social behaviors.

Dissociable effects of social context on song and doublecortin immunoreactivity in male canaries.

Variation in environmental factors such as day length and social context greatly affects reproductive behavior and the brain areas that regulate these behaviors. One such behavior is song in songbirds, which males use to attract a mate during the breeding season. In these species the absence of a potential mate leads to an increase in the number of songs produced, while the presence of a mate greatly diminishes singing. Interestingly, although long days promote song behavior, producing song itself can promote the incorporation of new neurons in brain regions controlling song output. Social context can also affect such neuroplasticity in these song control nuclei. The goal of the present study was to investigate in canaries (Serinus canaria), a songbird species, how photoperiod and social context affect song and the incorporation of new neurons, as measured by the microtubule-associated protein doublecortin (DCX) in HVC, a key vocal production brain region of the song control system. We show that long days increased HVC size and singing activity. In addition, male canaries paired with a female for 2 weeks showed enhanced DCX-immunoreactivity in HVC relative to birds housed alone. Strikingly, however, paired males sang fewer songs that exhibited a reduction in acoustic features such as song complexity and energy, compared with birds housed alone, which sang prolifically. These results show that social presence plays a significant role in the regulation of neural and behavioral plasticity in songbirds and can exert these effects in opposition to what might be expected based on activity-induced neurogenesis.

Differential effects of global versus local testosterone on singing behavior and its underlying neural substrate.

Steroid hormones regulate multiple but distinct aspects of social behaviors. Testosterone (T) has multiple effects on learned courtship song in that it regulates both the motivation to sing in a particular social context as well as the quality of song produced. The neural substrate(s) where T acts to regulate the motivation to sing as opposed to other aspects of song has not been definitively characterized. We show here that T implants in the medial preoptic nucleus (POM) of castrated male canaries (Serinus canaria) increase song rate but do not enhance acoustic features such as song stereotypy compared with birds receiving peripheral T that can act globally throughout the brain. Strikingly, T action in the POM increased song control nuclei volume, consistent with the hypothesis that singing activity induces neuroplasticity in the song control system independent of T acting in these nuclei. When presented with a female canary, POM-T birds copulated at a rate comparable to birds receiving systemic T but produced fewer calls and songs in her presence. Thus, POM is a key site where T acts to activate copulation and increase song rate, an appetitive sexual behavior in songbirds, but T action in other areas of the brain or periphery (e.g., HVC, dopaminergic cell groups, or the syrinx) is required to enhance the quality of song (i.e., stereotypy) as well as regulate context-specific vocalizations. These results have broad implications for research concerning how steroids act at multiple brain loci to regulate distinct sociosexual behaviors and the associated neuroplasticity.