Anatomical specificity of the action of testosterone in relation to the regulation of birdsong and the underlying neuroplasticity

One important goal of the field of behavioral neuroscience is to develop theories about the relative importance of different parts of the central and peripheral nervous systems in the modulation of behavior. Using canaries, a well-studied songbird, this thesis investigates the different levels at which testosterone acts to regulate song behavior and the underlying neuroplasticity. Given the integral role hormones play in the modulation of birdsong, the distinct sites at which androgen receptors are distributed throughout the songbird brain and periphery, and the distinct roles specific nuclei play in regulating different features of song, songbirds like canaries are especially amenable to such an investigation. The results of this thesis demonstrate testosterone’s role in the regulation of birdsong and neuroplasticity is pleiotropic: the regulation of specific song features, such as the motivation to sing versus song quality, and the neuroplasticity of the circuitry that regulates birdsong, is dependent on where and how (genomic versus non-genomic-like) in the songbird brain and periphery testosterone acts. For instance, testosterone action at the syrinx, the avian vocal organ, plays a different role in the regulation of birdsong compared to its central substrate, the song control system. Within the song control system, the action of testosterone within the sensorimotor region HVC (acronym is name) plays a different regulatory role of song than the motor region called the robust nucleus of the arcopallium. Many of these song features were regulated by the rapid actions of estrogens, a key product of the aromatization of testosterone in the regulation of birdsong. This suggests that estrogens are key in the regulation of song over very short time scales. Lastly, testosterone drove changes in plasticity in the song control system in a direct (acting within specific sites) as well as in an indirect (by enhancing singing activity) manner. This pleiotropic regulation by testosterone also applies to highly socio-sexually relevant vocal signals including in the songs called trills. These results highlight the complex role played by steroid hormones in the coordination of various suites of behaviors into a functional, adaptive response

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

There are no conflicts of interest to declare. This paper developed from the 2016 Early Career Impact Award from the Federation of Associations in Behavioral & Brain Sciences to TJS. TJS has received funding from The Leverhulme Trust. FJPE is in receipt of funding from the BBSRC (BB/M001555/1). The National Institutes of Health has funded RDF (NS 034950, NS093277, NIMH 087930), AGO (HD079573, IOS-1354760) and AMK (HD081959). BAA is an Arnold O. Beckman postdoctoral fellow.Peer reviewedPostprin

Androgen receptor alpha deficiency impacts aromatase expression in the female cichlid brain

Steroid hormones bind to specific receptors that act as transcription factors to modify gene expression in the brain to regulate physiological and behavioural processes. The specific genes controlled by steroid hormones in the brain are not fully known. Identifying these genes is integral to establishing a comprehensive understanding of how hormones impact physiology and behaviour. A popular organism for answering this question is the cichlid fish Astatotilapia burtoni. Recently, CRISPR/Cas9 was used to engineer A. burtoni that lack functional androgen receptor (AR) genes encoding ARα. ARα mutant male A. burtoni produced fewer aggressive displays and possessed reduced expression of the gene encoding brain-specific aromatase, cyp19a1, in the ventromedial hypothalamus (VMH), an aggression locus. As a follow-up, we investigated whether ARα deficiency affected cyp19a1 expression in female A. burtoni using the same genetic line. We find that female A. burtoni possessing one or two non-functional ARα alleles had much higher expression of cyp19a1 in the preoptic area (POA), while females with one non-functional ARα allele possessed lower expression of cyp19a1 in the putative fish homologue of the bed nucleus of the stria terminalis (BNST). Thus, ARα may have a sex-specific role in modifying cyp19a1 expression in the teleost POA and BNST, regions that underlie sex differences across vertebrates

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

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” dis- tinction 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 testos- terone 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 cor- related 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

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

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

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

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

Testosterone regulates birdsong in an anatomically specific manner

peer reviewedThe functions of birdsong include attracting a mate and repelling competitors. It is therefore not surprising that, in males in the temperate zone especially, birdsong is often produced in the context of reproduction. Testosterone of gonadal origin increases during the reproductive phase of the annual cycle and can significantly influence song production as well as song development via effects on song crystallization (testosterone secretion at the time of sexual maturity is essential for full crystallization to occur). In males, testosterone of gonadal origin can exert its effects in the brain on song and other reproductive behaviour via its oestrogenic and androgenic metabolites. The widespread distribution of nuclear androgen receptors in the song system and the occurrence of oestrogen receptors in one key forebrain nucleus in certain species as well as the presence of both receptor types in the diencephalon and the midbrain raises questions as to where and how testosterone is exerting its myriad effects on song. By selectively implanting testosterone into specific brain regions of castrated male canaries, Serinus canaria, we have identified the medial preoptic area as a critical site for the induction of a generalized increase in motivation that includes the motivation to sing. Testosterone action in the forebrain song nucleus HVC in contrast increases song stereotypy. Canaries receiving testosterone in the preoptic area and HVC sing stereotypic songs but at a much lower amplitude, indicating that testosterone's effects on amplitude are regulated elsewhere in the brain or the periphery. The lateral part of the magnocellular nucleus anterior nidopallium (LMAN) is a song nucleus that expresses a high density of androgen receptors and plays a role as a song variability generator during song learning. When adult female canaries are treated with testosterone, their song becomes more complex. Lesions to LMAN attenuate these effects on adult behavioural change, suggesting that testosterone may act in LMAN during song ontogeny to modulate changes associated with song crystallization. These anatomically specific effects illustrate how a single hormone can coordinate changes in many aspects of a complex behaviour to facilitate successful reproduction. © 2016 The Association for the Study of Animal BehaviourGrant from the National Institutes of Health NIH/NINDS R01 NS35467; BELSPO grant SSTC PAI P7/1