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Background. Behavioral sex differences are widespread in the animal world. These differences can be qualitative (i.e., behavior present in one sex but not the other, a true sex dimorphism) or quantitative (behavior is present at a higher rate or quality in one sex compared to the other). Singing in oscine songbirds is associated with both types of differences. In canaries, female rarely sing spontaneously but they can be induced to do so by treatments with steroids. Song in these females is however not fully masculinized and exhibits relatively subtle differences in quality as compared with male song. We analyzed here sex differences in syllable content and syllable use between singing male and female canaries. Methods. Songs were recorded from 3 groups of castrated male and 3 groups of photoregressed female canaries that had received Silastic™ implants filled with testosterone (T), with T plus estradiol (E2), or left empty (control). After 6 weeks of hormone treatment, 30 songs were recorded from each of the 47 subjects. Songs were segmented and each syllable was annotated. Various metrics of syllable diversity were extracted and network analysis was employed to characterize syllable sequences. Results. Male and female songs were characterized by marked sex differences related to syllable use. Compared to females, males had a larger syllable type repertoire and their songs contained more syllable types. Network analysis of syllable sequences showed that males follow more fixed patterns of syllable transitions than females. Both sexes however produced song of the same duration containing the same number of syllables produced at similar rates (numbers per second). Conclusions. Under the influence of T canaries of both sexes are able to produce generally similar vocalizations that nevertheless differ in specific ways. The development of song during ontogeny appears to be a very sophisticated process that is presumably based on genetic and endocrine mechanisms but also on specific learning processes. These data highlight the importance of detailed behavioral analyses in order to identify the many dimensions of a behavior that can differ between males and females.

The song control nucleus HVC of songbirds has emerged as a widespread model system to study adult neurogenesis and the factors that modulate the incorporation of new neurons, including seasonal state, sex differences or sex steroid hormone concentrations. However, the specific function of these new neurons born in adulthood remains poorly understood. We implemented a new procedure based on focal X-ray irradiation to deplete neural progenitors in the ventricular zone adjacent to HVC and study the functional consequences. A 23 Gy dose depleted by more than 50 percent the incorporation of BrdU in neural progenitors, a depletion that was confirmed by a significant decrease in doublecortin positive neurons. This depletion of neurogenesis significantly increased the variability of testosterone-induced songs in females and decreased their bandwidth. Expression of the immediate early gene ZENK (a.k.a. egr-1) in secondary auditory areas of the telencephalon that respond to song was also inhibited. These data provide compelling evidence that new neurons in HVC play a role in both song production and perception and that X-ray focal irradiation represents an excellent tool to advance our understanding of adult neurogenesis.

Birdsong has been the subject of broad research from a variety of sub-disciplines and has taught us much about the evolution, function, and mechanisms driving animal communication and cognition. Typically, birdsong refers to the specialized vocalizations produced by oscines. Historically, much of the research on birdsong was conducted in north temperate regions (specifically in Europe and North America) leading to multiple biases. Due to these historic biases these vocalizations are generally considered to be highly sexually dimorphic, heavily shaped by sexual selection and essential for courtship and territoriality. Song is also typically defined as a learned trait shaped by cultural evolution. Together, this framework focuses research specifically on males, particularly during the north temperate breeding season - reflecting and thereby reinforcing this framework. The physiological underpinnings of song often emphasize the role of the hypothalamic-pituitary-gonadal axis (associated with breeding changes) and the song control system (underlying vocal learning). Over the years there has been great debate over which features of song are essential to the definition of birdsong, which features apply broadly to contexts outside males in the north temperate region, and over the importance of having a definition at all. Importantly, the definitions we use can both guide and limit the progress of research. Here, we describe the history of these definitions, and how these definitions have directed and restricted research to focus on male song in sexually selected contexts. Additionally, we highlight the gaps in our scientific knowledge, especially with respect to the function and physiological mechanisms underlying song in females and in winter, as well as in non-seasonally breeding species. Furthermore, we highlight the problems with using complexity and learning as dichotomous variables to categorize songs and calls. Across species, no one characteristic of song - sexual dimorphism, seasonality, complexity, sexual selection, learning - consistently delineates song from other songbird vocal communication. We provide recommendations for next steps to build an inclusive information framework that will allow researchers to explore nuances in animal communication and promote comparative research. Specifically, we recommend that researchers should operationalize the axis of variation most relevant to their study/species by identifying their specific question and the variable(s) of focus (e.g. seasonality). Researchers should also identify the axis (axes) of variation (e.g. degree of control by testosterone) most relevant to their study and use language consistent with the question and axis (axes) of variation (e.g. control by testosterone in the seasonal vocal production of birds).

Studies of acoustic communication often focus on the categories and units of vocalizations, but subtle variation also occurs in how these signals are uttered. In human speech, it is not only phonemes and words that carry information but also the timbre, intonation, and stress of how speech sounds are delivered (often referred to as “paralinguistic content”). In non-human animals, variation across utterances of vocal signals also carries behaviorally relevant information across taxa. However, the discriminability of these cues has been rarely tested in a psychophysical paradigm. Here, we focus on acoustic communication in the zebra finch (Taeniopygia guttata), a songbird species in which the male produces a single stereotyped motif repeatedly in song bouts. These motif renditions, like the song repetitions of many birds, sound very similar to the casual human listener. In this study, we show that zebra finches can easily discriminate between the renditions, even at the level of single song syllables, much as humans can discriminate renditions of speech sounds. These results support the notion that sensitivity to fine acoustic details may be a primary channel of information in zebra finch song, as well as a shared, foundational property of vocal communication systems across species.

The avian homologue of oxytocin (OT), formerly called mesotocin, influences social behaviors in songbirds and potentially song production. We sought to characterize the distribution of OT peptide in the brain of two songbird species: canaries (Serinus canaria) and zebra finches (Taeniopygia guttata). To visualize OT, we performed immunocytochemistry using an antibody previously shown to identify OT in avian species. In both canaries and zebra finches, dense OT-ir perikarya were located in the paraventricular nucleus (PVN), preoptic area (POA), supraoptic nucleus (SON), and medial bed nucleus of the stria terminalis (BNSTm). We also observed morphologically distinct OT-ir cells scattered throughout the mesopallium. OT-ir fibers were observed in the PVN, ventral medial hypothalamus (VMH), periaqueductal gray (PAG), intercollicular nucleus (ICo), and ventral tegmental area (VTA). We also observed punctate OT-ir fibers in the song control nucleus HVC. In both male and female canaries, OT-ir fibers were present in the lateral septum (LS), but innervation was greater in males. We did not observe this sex difference in zebra finches. Much of the OT staining observed is consistent with general distributions within the vertebrate hypothalamus, indicating a possible conserved function. However, some extra-hypothalamic distributions, such as perikarya in the mesopallium, may be specific to songbirds and play a role in song perception and production. The presence of OT-ir fibers in HVC and song control nuclei projecting dopaminergic regions provides anatomical evidence in support of the idea that OT can influence singing behavior-either directly via HVC or indirectly via the PAG, VTA, or POA.

Testosterone aromatization into estrogens in the preoptic area (POA) is critical for the activation of male sexual behavior in many vertebrates. Yet, the cellular mechanisms mediating actions of neuroestrogens on sexual behavior remain largely unknown. We investigated in male and female Japanese quail by dual-label fluorescent in situ hybridization (FISH) whether aromatase-positive (ARO) neurons express glutamic acid decarboxylase 67 (GAD67), the rate-limiting enzyme in GABA biosynthesis. ARO cells and ARO cells double labeled with GAD67 (ARO-GAD67) were counted at standardized locations in the medial preoptic nucleus (POM) and the medial bed nucleus of the stria terminalis (BST) to produce three-dimensional distribution maps. Overall, males had more ARO cells than females in POM and BST. The number of double-labeled ARO-GAD67 cells was also higher in males than in females and greatly varied as a function of the specific position in these nuclei. Significant sex differences were however present only in the most caudal part of POM. Although both ARO and GAD67 were expressed in the VMN, no colocalization between these markers was detected. Together, these data show that a high proportion of estrogen-synthesizing neurons in POM and BST are inhibitory and the colocalization of GAD67 with ARO exhibits a high degree of anatomical specificity as well as localized sex differences. The fact that many preoptic ARO neurons project to the periaqueductal gray in male quail suggests possible mechanisms through which locally produced estrogens could activate male sexual behavior.

Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing toward an active role of thyroid hormones during this window of possible neuroplasticity. In contrast, DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Both under natural conditions as with methimazole treatment, circulating thyroid hormone levels decreased during the photosensitive period, which coincided with the onset of neuroplasticity. This inverse relationship between thyroid hormones and neuroplasticity was further demonstrated by the negative correlation between plasma T3 and the microstructural changes in several song control nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the photostimulated period inhibited the increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic-pituitary-gonadal (HPG) axis. The lack of high testosterone levels influenced the song behavior of hypothyroid starlings, while the lack of high plasma T4 during photostimulation affected the myelination of several tracts. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake on neuroplasticity imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Whereas, an increase in circulating T4 during the photostimulated period potentially influences the myelination of several white matter tracts, which stabilizes the neuroplastic changes. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity. ispartof: FRONTIERS IN MOLECULAR NEUROSCIENCE vol:15 ispartof: location:Switzerland status: published

Birdsong is controlled in part by a discrete network of interconnected brain nuclei regulated in turn by steroid hormones and environmental stimuli. This complex interaction results in neural changes that occur seasonally as the environment varies (e.g., photoperiod, food/water availability, etc.). Variation in environment, vocal behavior, and neuroendocrine control has been primarily studied in male songbirds in both laboratory studies of captive birds and field studies of wild caught birds. The bias toward studying seasonality in the neuroendocrine regulation of song in male birds comes from a historic focus on sexually selected male behaviors. In fact, given that male song is often loud and accompanied by somewhat extravagant courtship behaviors, female song has long been overlooked. To compound this bias, the primary model songbird species for studies in the lab, zebra finches (Taeniopygia guttata) and canaries (Serinus canaria), exhibit little or no female song. Therefore, understanding the degree of variation and neuroendocrine control of seasonality in female songbirds is a major gap in our knowledge. In this review, we discuss the importance of studying sex differences in seasonal plasticity and the song control system. Specifically, we discuss sex differences in 1) the neuroanatomy of the song control system, 2) the distribution of receptors for androgens and estrogens and 3) the seasonal neuroplasticity of the hypothalamo-pituitary-gonadal axis as well as in the neural and cellular mechanisms mediating song system changes. We also discuss how these neuroendocrine mechanisms drive sex differences in seasonal behavior. Finally, we highlight specific gaps in our knowledge and suggest experiments critical for filling these gaps.

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.

In vertebrates, advanced cognitive abilities are typically associated with the telencephalic pallium. In mammals, the pallium is a layered mixture of excitatory and inhibitory neuronal populations with distinct molecular, physiological, and network phenotypes. This cortical architecture is proposed to support efficient, high-level information processing. Comparative perspectives across vertebrates provide a lens to understand the common features of pallium that are important for advanced cognition. Studies in songbirds have established strikingly parallel features of neuronal types between mammalian and avian pallium. However, lack of genetic access to defined pallial cell types in non-mammalian vertebrates has hindered progress in resolving connections between molecular and physiological phenotypes. A definitive mapping of the physiology of pallial cells onto their molecular identities in birds is critical for understanding how synaptic and computational properties depend on underlying molecular phenotypes. Using viral tools to target excitatory vs. inhibitory neurons in the zebra finch auditory association pallium (CaMKIIα and GAD1 promoters respectively), we systematically tested predictions derived from mammalian pallium. We identified two genetically-distinct neuronal populations that exhibit profound physiological and computational similarities with mammalian excitatory and inhibitory pallial cells, definitively aligning putative cell types in avian caudal nidopallium with these molecular identities. Specifically, genetically-identified CaMKIIα and GAD1 cell types in avian auditory association pallium exhibit distinct intrinsic physiological parameters, distinct auditory coding principles, and inhibitory-dependent pallial synchrony, gamma oscillations, and local suppression. The retention or convergence of these molecular and physiological features in both birds and mammals clarifies the characteristics of pallial circuits for advanced cognitive abilities.

Belgian Waterslager song canaries, bred for hundreds of years for a low-pitched song, have also acquired an inherited high-frequency hearing loss associated with hair cell abnormalities. Here, auditory thresholds measured using auditory brainstem responses and psychophysical methods in three different strains of canaries are compared: Belgian Waterslagers, American Singers, and Borders. Border canaries have not been bred for song characteristics while American Singer canaries have been bred for song only since the 1930s. Results show that American Singer canaries also have elevated high frequency thresholds that are similar to those of the Belgian Waterslager, while Border canaries have normal thresholds. These results strengthen the case that song canary breeders in selecting for song characteristics may have inadvertently selected for hearing abnormalities.

Female songbirds are thought to make mate choices based on aspects of male song quality. Male canaries (Serinus canaria) produce songs with "special" syllables that have been shown to be highly salient to female listeners - eliciting high rates of sexual displays and enhanced immediate early gene (IEG) expression. Immunohistochemistry for the IEG ZENK was used to examine the effects of experience with these syllables on activity in the caudal mesopallium (CMM) and nidocaudal mesopallium (NCM), two auditory areas important in processing conspecific song. Photostimulated female canaries were housed in sound attenuated chambers and played pseudosongs containing either three special syllables or three non-special syllables, an intro, and an outro sequence. Females that heard special syllable pseudosongs exhibited higher ZENK expression in CMM. To assess the effects of experience, photostimulated females were pair housed and exposed to playback of song with or without special syllables for 14 days. After transfer to individual housing, birds were played one of the aforementioned stimuli or silence. ZENK expression in CMM and NCM was equivalent for song with and without special syllables, but significantly lower for silence. Females who experienced song with special syllables had lower plasma estradiol concentrations after final song playback. This study indicates that CMM exhibits an IEG response bias to special syllables in limited acoustic contexts, but not in full song, which may contain additional biologically relevant information. Furthermore, estradiol concentrations may mediate changes in song responses, serving as a mechanism for modulating mate choice in differing song environments.

Testosterone activates singing within days in castrated male songbirds but full song quality only develops after a few weeks. Lesions of the medial preoptic nucleus (POM) inhibit while stereotaxic testosterone implants into this nucleus increase singing rate suggesting that this site plays a key role in the regulation of singing motivation. Testosterone action in the song control system works in parallel to control song quality. Accordingly, systemic testosterone increases POM volume within 1–2 days in female canaries, while the increase in volume of song control nuclei takes at least 2 weeks. The current study tested whether testosterone action is associated with similar differences in latencies in males. Photosensitive castrated male canaries were implanted with testosterone-filled Silastic™ implants and control castrates received empty implants, while simultaneously the photoperiod was switched from short- to long-days. Brains were collected from all subjects two days later. Plasma testosterone was elevated in testosterone-treated but not in controls. HVC volumes were not affected, but testosterone significantly increased the POM volume as identified by the dense group of aromatase-immunoreactive neurons, the number and somal area of these neurons and the fractional area they cover in POM. Testosterone-treated females from a previous experiment had a smaller POM volume in similar conditions suggesting the existence of a stable sex difference potentially affecting singing behavior. Thus testosterone induces male POM growth and aromatase expression in this nucleus within two days without affecting HVC size, further supporting the notion that testosterone increases singing motivation via its action in POM.

This study was designed to determine whether changes in sexual motivation acutely regulate brain estrogen synthesis by aromatase. Five experiments (Exp.1–5) were first conducted to determine the effect of recent mating and of the presentation of a new female (Coolidge effect) on sexual motivation. Exp.1–2 showed that 10 min or overnight access to copulation decreases measures of male sexual motivation when male subjects were visually exposed to the female they had copulated with and this effect is not counteracted by the view of a new female. Exp.3 showed that sexual motivation is revived by the view of a new female in previously unmated males only allowed to see another female for 10 min. After mating for 10 min (Exp.4) or overnight (Exp.5) with a female, males showed a decrease in copulatory behavior that was not reversed by access to a new female. Exp.6 and 7 confirmed that overnight copulation (Exp.6) and view of a novel female (Exp.7) respectively decreases and increases sexual behavior and motivation. Yet, these manipulations did not affect brain aromatase activity except in the tuberal hypothalamus. Together these data confirm that copulation or prolonged view of a female decrease sexual motivation but a reactivation of sexual motivation by a new female can only be obtained if males had only seen another female but not copulated with her, which is different in some degree from the Coolidge effect described in rodents. Moreover changes in brain aromatase do not simply reflect changes in motivation and more complex mechanisms must be considered.

Aromatization within the medial preoptic nucleus (POM) is essential for the expression of male copulatory behavior in Japanese quail. However, several nuclei within the social behavior network (SBN) also express aromatase. Whether aromatase in these loci participates in the behavioral activation is not known. Castrated male Japanese quail were implanted with 2 subcutaneous Silastic capsules filled with crystalline testosterone and with bilateral stereotaxic implants filled with the aromatase inhibitor Vorozole targeting the POM, the bed nucleus of the stria terminalis (BST) or the ventromedial nucleus of the hypothalamus (VMN). Control animals were implanted with testosterone and empty bilateral stereotaxic implants. Starting 2 days after the surgery, subjects were tested for the expression of consummatory sexual behavior (CSB) every other day for a total of 10 tests. They were also tested once for appetitive sexual behavior (ASB) as measured by the rhythmic cloacal sphincter movements displayed in response to the visual presentation of a female. CSB was drastically reduced when the Vorozole implants were localized in the POM, but not in the BST nor in the VMN. Birds with implants in the BST showed a longer latency to show CSB in the first 6 tests than controls, suggesting a role of the BST in the acquisition of the full copulatory ability. ASB was not significantly affected by aromatase blockade in any region. These data confirm the key role played by the POM in the control of male sexual behavior and suggest a minor role for aromatization in the BST or VMN.

Temperate-zone birds display marked seasonal changes in reproductive behaviors and the underlying hormonal and neural mechanisms. These changes were extensively studied in canaries (Serinus canaria) but differ between strains. Fife fancy male canaries change their reproductive physiology in response to variations in day length but it remains unclear whether they become photorefractory (PR) when exposed to long days and what the consequences are for gonadal activity, singing behavior and the associated neural plasticity. Photosensitive (PS) male birds that had become reproductively competent (high song output, large testes) after being maintained on short days (SD, 8 L:16D) for 6 months were divided into two groups: control birds remained on SD (SD-PS group) and experimental birds were switched to long days (16 L:8D) and progressively developed photorefractoriness (LD-PR group). During the following 12 weeks, singing behavior (quantitatively analyzed for 3 × 2 hours every week) and gonadal size (repeatedly measured by CT X-ray scans) remained similar in both groups but there was an increase in plasma testosterone and trill numbers in the LD-PR group. Day length was then decreased back to 8 L:16D for LD-PR birds, which immediately induced a cessation of song, a decrease in plasma testosterone concentration, in the volume of song control nuclei (HVC, RA and Area X), in HVC neurogenesis and in aromatase expression in the medial preoptic area. These data demonstrate that Fife fancy canaries readily respond to changes in photoperiod and display a pattern of photorefractoriness following exposure to long days that is associated with marked changes in brain and behavior.

Adult treatments with testosterone (T) do not activate singing behavior nor promote growth of song control nuclei to the same extent in male and female canaries (Serinus canaria). Because T acts in part via aromatization into an estrogen and brain aromatase activity is lower in females than in males in many vertebrates, we hypothesized that this enzymatic difference might explain the sex differences seen even after exposure to the same amount of T. Three groups of castrated males and 3 groups of photoregressed females (i.e., with quiescent ovaries following exposure to short days) received either 2 empty 10 mm silastic implants, one empty implant and one implant filled with T or one implant filled with T plus one with estradiol (E2). Songs were recorded for 3 h each week for 6 weeks before brains were collected and song control nuclei volumes were measured in Nissl-stained sections. Multiple measures of song were still different in males and females following treatment with T. Co-administration of E2 did not improve these measures and even tended to inhibit some measures such as song rate and song duration. The volume of forebrain song control nuclei (HVC, RA, Area X) and the rate of neurogenesis in HVC was increased by the two steroid treatments, but remained significantly smaller in females than in males irrespective of the endocrine condition. These sex differences are thus not caused by a lower aromatization of the steroid; sex differences in canaries are probably organized either by early steroid action or by sex-specific gene regulation directly in the brain.

Male song in songbirds is a critical and elaborate signal for mate attraction. In many species female listeners respond to male song both behaviorally and physiologically (e.g. copulation solicitation displays and production of the immediate early gene ZENK in auditory regions). It is becoming increasingly well known that females in many species also sing. However, in common lab species, such as canaries (Serinus canaria), female song is limited and has been primarily studied in the context of administering of exogenous testosterone (T) to increase song rate and length. In this study we addressed to what extent female canary songs are masculinized by the administration of exogenous T based on the behavioral and physiological responses of avian receivers. Specifically, are T induced female songs sufficient to elicit courtship behaviors and auditory ZENK expression in female listeners? We found that female songs after 3 weeks of exogenous T were significantly longer and more complex than female songs after 12 weeks of exogenous T. Additionally, we found that playback of 3-week T song significantly increased sexual response behaviors and the expression of ZENK in the auditory brain regions of female listeners. Finally, we conclude that extended periods of T do not necessarily maintain the masculinization of female song.

Songbirds learn their vocalizations during developmental sensitive periods of song memorization and sensorimotor learning. Some seasonal songbirds, called open-ended learners, recapitulate transitions from sensorimotor learning and song crystallization on a seasonal basis during adulthood. In adult male canaries, sensorimotor learning occurs each year in autumn and leads to modifications of the syllable repertoire during successive breeding seasons. We previously showed that perineuronal nets (PNN) expression in song control nuclei decreases during this sensorimotor learning period. Here we explored the causal link between PNN expression in adult canaries and song modification by enzymatically degrading PNN in HVC, a key song control system nucleus. Three independent experiments identified limited effects of the PNN degradation in HVC on the song structure of male canaries. They clearly establish that presence of PNN in HVC is not required to maintain general features of crystallized song. Some suggestion was collected that PNN are implicated in the stability of song repertoires but this evidence is too preliminary to draw firm conclusions and additional investigations should consider producing PNN degradations at specified time points of the seasonal cycle. It also remains possible that once song has been crystallized at the beginning of the first breeding season, PNN no longer play a key role in determining song structure which could be tested by treatments with ChABC at key step in ontogeny. It would in this context be important to develop multiple stereotaxic procedures allowing the simultaneous bilateral degradation of PNN in several song control nuclei for extended periods.

A species’ success during the invasion of new areas hinges on an interplay between demographic processes and the outcome of localized selection. Invasive European Starlings (Sturnus vulgaris) established populations in Australia and North America in the 19th century. Here, we compare whole-genome sequences among native and independently introduced European Starling populations from three continents to determine how demographic processes interact with rapid adaptive evolution to generate similar genetic patterns in these recent and replicated invasions. Our results confirm that a post-bottleneck expansion may in fact support local adaptation. We find that specific genomic regions have differentiated even on this short evolutionary timescale, and suggest that selection best explains differentiation in at least two of these regions. This infamous and highly mobile invader adapted to novel selection (e.g., extrinsic factors), perhaps in part due to the demographic boom intrinsic to many invasions.

The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second North American genome (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterisation. S. vulgaris vAU combined 10x Genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 1,628 scaffolds (72.5 Mb scaffold N50). Species-specific transcript mapping and gene annotation revealed high structural and functional completeness (94.6% BUSCO completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Rapid, recent advances in sequencing technologies and bioinformatics software have highlighted the need for evidence-based assessment of assembly decisions on a case-by-case basis. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, SAAGA) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counter-intuitive behaviour in traditional BUSCO metrics, and present Buscomp, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. Finally, we present a second starling assembly, S. vulgaris vNA, to facilitate comparative analysis and global genomic research on this ecologically important species.

Aromatase converts androgens into estrogens in the brain of vertebrates including humans. This enzyme is also expressed in other tissues where its action may result in negative effects on human health (e.g., promotion of tumor growth). To prevent these effects, aromatase inhibitors were developed and are currently used to block human estrogen-dependent tumors. In vertebrates including quail, aromatase is expressed in a highly conserved set of interconnected brain nuclei known as the social behavior network. This network is directly implicated in the expression of a large range of social behaviors. The primary goal of this study was to characterize in Japanese quail the potential impact of brain aromatase on sexual behavior, aggressiveness and social motivation (i.e., tendency to approach and stay close to conspecifics). An additional goal was to test the feasibility and effectiveness of long-term delivery of an aromatase inhibitor directly into the third ventricle via Alzet™ osmotic minipumps using male sexual behavior as the aromatase dependent measure. We demonstrate that this mode of administration results in the strongest inhibition of both copulatory behavior and sexual motivation ever observed in this species, while other social behaviors were variably affected. Sexual motivation and the tendency to approach a group of conspecifics including females clearly seem to depend on brain aromatase, but the effects of central estrogen production on aggressive behavior and on the motivation to approach males remain less clear.

In the 1970s, Nottebohm and Arnold reported marked male-biased sex differences in the volume of three song control nuclei in songbirds. Subsequently a series of studies on several songbird species suggested that there is a positive correlation between the degree to which there is a sex difference in the volume of these song control nuclei and in song behavior. This correlation has been questioned in recent years. Furthermore, it has become clear that the song circuit is fully integrated into a more comprehensive neural circuit that regulates multiple courtship and reproductive behaviors including song. Sex differences in songbirds should be evaluated in the context of the full complement of behaviors produced by both sexes in relation to reproduction and based on the entire circuit in order to understand the functional significance of variation between males and females in brain and behavior. Variation in brain and behavior exhibited among living songbird species provides an excellent opportunity to understand the functional significance of sex differences related to social behaviors.

Synopsis Research on monogamy has largely focused on marked behaviors that are unique to pair bonded partners. However, these marked behaviors represent only a subset of the pair-directed behaviors that partners engage in; the influence of pair bonding on mundane or subtle social interactions among partners remains largely unknown. In this study, we describe the changes that occur during brief social reunions (or greets) over the course of pair bonding in zebra finches. We quantified pair-directed behavior during 5-min reunions from three stages of pair bonding: initial pairing (between 4 and 72 h), early pairing (1–2 weeks), and late pairing (>1 month). These social interactions were operationalized in multiple ways. First, we quantified the overall activity levels (call and movement rates) for both the male and female. Overall, females were more active than males, but for both males and females calling activity was highest at initial pairing. We quantified behavioral coordination between partners in two ways: (1) similarity in call and movement rates between partners and (2) temporal synchrony of calls and movements between partners (via sliding correlation coefficients of time-stamped calls and movements). Overall, there were no effects of pairing stage on behavioral coordination. Finally, we used principal component analyses to disentangle behavioral coordination from the activity levels of the male and female. These results contribute to a growing line of evidence that male and female zebra finches differentially contribute to social dynamics and highlight the influence of pair bonding on the development of social dynamics. Furthermore, our preliminary analyses raise the hypothesis that behavioral coordination during the earliest phases of pairing is modulated by the extent and nature of prior experience. Overall, while behavioral coordination is clearly important for many salient interactions such as duetting, courtship displays, and biparental care, the significance of mundane social interactions for monogamous partnerships remains largely unknown.

Perineuronal nets (PNN) of the extracellular matrix are dense aggregations of chondroitin-sulfate proteoglycans that usually surround fast-spiking parvalbumin-expressing inhibitory interneurons (PV). The development of PNN around PV appears specifically at the end of sensitive periods of visual learning and limits the synaptic plasticity in the visual cortex of mammals. Seasonal songbirds display a high level of adult neuroplasticity associated with vocal learning, which is regulated by fluctuations of circulating testosterone concentrations. Seasonal changes in testosterone concentrations and in neuroplasticity are associated with vocal changes between the non-breeding and breeding seasons. Increases in blood testosterone concentrations in the spring lead to the annual crystallization of song so that song becomes more stereotyped. Here we explore whether testosterone also regulates PNN expression in the song control system of male and female canaries. We show that, in both males and females, testosterone increases the number of PNN and of PV neurons in the three main telencephalic song control nuclei HVC, RA (nucleus robustus arcopallialis) and Area X and increases the PNN localization around PV interneurons. Singing activity was recorded in males and quantitative analyses demonstrated that testosterone also increased male singing rate, song duration and song energy while decreasing song entropy. Together, these data suggest that the development of PNN could provide the synaptic stability required to maintain the stability of the testosterone-induced crystallized song. This provides the new evidence for a role of PNN in the regulation of adult seasonal plasticity in seasonal songbirds.

Contribution to Special Issue on Fast effects of steroids. Estrogens exert pleiotropic effects on multiple physiological and behavioral traits including sexual behavior. These effects are classically mediated via binding to nuclear receptors and subsequent regulation of target gene transcription. Estrogens also affect neuronal activity and cell-signaling pathways via faster, membrane-initiated events. Although the distinction between appetitive and consummatory aspects of sexual behavior has been criticized, this distinction remains valuable in that it facilitates the causal analysis of certain behavioral systems. Effects of neuroestrogens produced by neuronal aromatization of testosterone on copulatory performance (consummatory aspect) and on sexual motivation (appetitive aspect) are described in male quail. The central administration of estradiol rapidly increases expression of sexual motivation, as assessed by two measures of sexual motivation produced in response to the visual presentation of a female but not sexual performance in male Japanese quail. This effect is mimicked by membrane-impermeable analogs of estradiol, indicating that it is initiated at the cell membrane. Conversely, blocking the action of estrogens or their synthesis by a single intracerebroventricular injection of estrogen receptor antagonists or aromatase inhibitors, respectively, decreases sexual motivation within minutes without affecting performance. The same steroid has thus evolved complementary mechanisms to regulate different behavioral components (motivation vs. performance) in distinct temporal domains (long- vs. short-term) so that diverse reproductive activities can be properly coordinated. Changes in preoptic aromatase activity and estradiol as well as glutamate concentrations are observed during or immediately after copulation. The interaction between these neuroendocrine/neurochemical changes and their functional significance is discussed.

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.

In seasonally breeding songbirds such as canaries, singing behavior is predominantly under the control of testosterone and its metabolites. Short daylengths in the fall that break photorefractoriness are followed by increasing daylengths in spring that activate singing via both photoperiodic and hormonal mechanisms. However, we observed in a group of castrated male Fife fancy canaries maintained for a long duration under a short day photoperiod a large proportion of subjects that sang at high rates. This singing rate was not correlated with variation in the low circulating concentrations of testosterone. Treatment of these actively singing castrated male canaries with a combination of an aromatase inhibitor (ATD) and an androgen receptor blocker (flutamide) only marginally decreased this singing activity as compared to control untreated birds and did not affect various measures of song quality. The volumes of HVC and of the medial preoptic nucleus (POM) were also unaffected by these treatments but were relatively large and similar to volumes in testosterone-treated males. In contrast, peripheral androgen-sensitive structures such as the cloacal protuberance and syrinx mass were small, similar to what is observed in castrates. Together these data suggest that after a long-term steroid deprivation singing behavior can be activated by very low concentrations of testosterone. Singing normally depends on the activation by testosterone and its metabolites of multiple downstream neurochemical systems such as catecholamines, nonapeptides or opioids. These transmitter systems might become hypersensitive to steroid action after long term castration as they probably are at the end of winter during the annual cycle in seasonally breeding temperate zone species.

There is a rich history of behavioral and neurobiological research focused on the 'syntax' of birdsong as a model for human language and complex auditory perception. Zebra finches are one of the most widely studied songbird species in this area of investigation. As they produce song syllables in a fixed sequence, it is reasonable to assume that adult zebra finches are also sensitive to the order of syllables within their song; however, results from electrophysiological and behavioral studies provide somewhat mixed evidence on exactly how sensitive zebra finches are to syllable order as compared, say, to syllable structure. Here, we investigate how well adult zebra finches can discriminate changes in syllable order relative to changes in syllable structure in their natural song motifs. In addition, we identify a possible role for experience in enhancing sensitivity to syllable order. We found that both male and female adult zebra finches are surprisingly poor at discriminating changes to the order of syllables within their species-specific song motifs, but are extraordinarily good at discriminating changes to syllable structure (i.e., reversals) in specific syllables. Direct experience or familiarity with a song, either using the bird's own song (BOS) or the song of a flock mate as the test stimulus, improved both male and female zebra finches' sensitivity to syllable order. However, even with experience, birds remained much more sensitive to structural changes in syllables. These results help to clarify some of the ambiguities from the literature on the discriminability of changes in syllable order in zebra finches, provide potential insight on the ethological significance of zebra finch song features, and suggest new avenues of investigation in using zebra finches as animal models for sequential sound processing.

Numerous studies have evaluated changes in time of testicular development in birds by exploratory laparotomy or post-mortem autopsy. The invasive nature of these approaches has obviously limited the frequency at which these measures can be collected. We demonstrate here that accurate assessment of gonadal size can be reliably and repeatedly obtained by computer-assisted X-ray tomography (CT scans). This approach provides images of the testes in the three orthogonal planes that allow measuring either the largest diameter or even the volume of the testes, providing results that match those obtained by surgical approaches.

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.

Estrogens are known to act rapidly, probably via membrane estrogen receptors, to induce fast effects on physiological and behavioral processes. Engaging in some of these behaviors, such as sexual behavior, results in an acute modulation of the production of estrogens in the brain by regulating the efficiency of the estrogen synthase enzyme, aromatase. Specifically, we recently demonstrated that aromatase activity (AA) in the male quail brain is rapidly inhibited in discrete brain regions including the medial preoptic nucleus (POM) following exposure to a female. Evidence from in vitro studies point to glutamate release as one of the mechanisms controlling these rapid regulations of the aromatase enzyme. Here, we show that (a) the acute injection of the glutamatergic agonist kainate into the POM of anesthetized male quail inhibits AA and (b) glutamate is released in the POM during copulation. These results provide the first set of in vivo data demonstrating a role for glutamate release in the rapid control of AA in the context of sexual behavior.

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience-dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open-ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin-positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed-ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin-positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed-ended and open-ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975-994, 2017.

The 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.

The complex and melodic nature of many birds' songs has raised interest in potential parallels between avian vocal sequences and human speech. The similarities between birdsong and speech in production and learning are well established, but surprisingly little is known about how birds perceive song sequences. One popular laboratory songbird, the zebra finch ( Taeniopygia guttata ), has recently attracted attention as an avian model for human speech, in part because the male learns to produce the individual elements in its song motif in a fixed sequence. But psychoacoustic evidence shows that adult zebra finches are relatively insensitive to the sequential features of song syllables. Instead, zebra finches and other birds seem to be exquisitely sensitive to the acoustic details of individual syllables to a degree that is beyond human hearing capacity. Based on these findings, we present a finite-state model of zebra finch perception of song syllable sequences and discuss the rich informational capacity of their vocal system. Furthermore, we highlight the abilities of budgerigars ( Melopsittacus undulatus ), a parrot species, to hear sequential features better than zebra finches and suggest that neurophysiological investigations comparing these species could prove fruitful for uncovering neural mechanisms for auditory sequence perception in human speech. This article is part of the theme issue ‘What can animal communication teach us about human language?’

An individual's ability to respond to and align with the behavior of others is a fundamental component of social behavior. Zebra finches form lifelong monogamous pair bonds; however, zebra finches are also gregarious and can form strong social bonds with same-sex conspecifics. Here, we quantified behavior during brief 10-min reunions for males and females in five types of social conditions: monogamously bonded opposite-sex partners, familiar same-sex, familiar opposite-sex, novel same-sex, and novel opposite-sex dyads. We analyzed these interactions in three ways. First, we quantified overall activity levels (call and movement rates) for each individual. Second, we measured how coordinated calls and movements were by calculating (a) the percent difference in activity rates as an estimate of how similar calling and movement activity were between individuals within a dyad, and (b) the sliding correlation coefficients for time-stamped calls and movements for each dyad. Finally, we described multimodal behavioral profiles of coordination using principal component analyses. Overall, females were more active than males. For both females and males, activity levels as well as the coordination of calls and movements were significantly affected by social condition. In general, monogamous partners, female familiar same-sex dyads and familiar opposite-sex dyads were the most coordinated. This effect of familiarity shows that moment-to-moment behavioral coordination can be influenced by prior social experiences. Quantifying patterns of coordination or social synchrony may prove valuable for understanding the effects of social experience on brain and behavior. (PsycInfo Database Record (c) 2020 APA, all rights reserved).