Estrogen contributes to seasonal plasticity of the adult avian song control system

Songbirds show dramatic neural plasticity as adults, including large-scale anatomical changes in discrete brain regions ("song control nuclei") controlling the production of singing behavior. The volumes of several song control nuclei are much larger in the breeding season than in the nonbreeding season, and these seasonal neural changes are regulated by plasma testosterone (T) levels. In many cases, the effects of T on the central nervous system are mediated by neural conversion to estradiol (E(2)) by the enzyme aromatase. The forebrain of male songbirds expresses very high levels of aromatase, in some cases adjacent to song control nuclei. We examined the effects of aromatase inhibition and estrogen treatment on song nuclei size using wild male songbirds in both the breeding and nonbreeding seasons. In breeding males, aromatase inhibition caused the volume of a telencephalic song control nucleus (HVC) to decrease, and this effect was partially rescued by concurrent estrogen replacement. In nonbreeding males, estradiol treatment caused HVC to grow to maximal spring size within 2 weeks. Overall, these data suggest that aromatization of T is an important mediator of song control system plasticity, and that estradiol has neurotrophic effects in adult male songbirds. This study demonstrates that estrogen can affect adult neural plasticity on a gross anatomical scale and is the first examination of estrogen effects on the brain of a wild animal.     

A species-specific view of song representation in a sensorimotor nucleus

Songbirds constitute a powerful model system for the investigation of how complex vocal communication sounds are represented and generated, offering a neural system in which the brain areas involved in auditory, motor and auditory–motor integration are well known. One brain area of considerable interest is the nucleus HVC. Neurons in the HVC respond vigorously to the presentation of the bird’s own song and display song-related motor activity. In the present paper, we present a synthesis of neurophysiological studies performed in the HVC of one songbird species, the canary (Serinus canaria). These studies, by taking advantage of the singing behavior and song characteristics of the canary, have examined the neuronal representation of the bird’s own song in the HVC. They suggest that breeding cues influence the degree of auditory selectivity of HVC neurons for the bird’s own song over its time-reversed version, without affecting the contribution of spike timing to the information carried by these two song stimuli. Also, while HVC neurons are collectively more responsive to forward playback of the bird’s own song than to its temporally or spectrally modified versions, some are more broadly tuned, with an auditory responsiveness that extends beyond the bird’s own song. Lastly, because the HVC is also involved in song production, we discuss the peripheral control of song production, and suggest that interspecific variations in song production mechanisms could be exploited to improve our understanding of the functional role of the HVC in respiratory–vocal coordination.     

Aromatase inhibition affects testosterone-induced masculinization of song and the neural song system in female canaries

Songbirds have a specialized steroid-sensitive network of brain nuclei, the song system, for controlling song. Most nuclei of the song system express androgen receptors, and the sensory-motor integration nucleus High Vocal Center (HVC) alone also expresses estrogen receptors. Apart from expressing estrogen receptors in the vocal control system, songbirds are unique among birds because they have high concentrations of the estrogen-synthesizing enzyme aromatase in the neostriatum surrounding HVC. However, the role of estrogen in controlling the development of the song structure has been scarcely investigated. In this work, we show that blocking the production of estrogen during testosterone-induced song motor development in adult female canaries alters the song pattern compared to control females treated with testosterone only. These effects were correlated with inhibition of the expression of estrogen-sensitive genes, such as brain-derived nerve growth factor, in HVC. The expression of the ATP-synthase gene, an indicator of cell activity, in HVC, and the size of HVC, were not affected by the treatment. Our results provide the first example of estrogen-sensitive mechanisms controlling the structural features of adult birdsong.     

Aromatase inhibition affects testosterone‐induced masculinization of song and the neural song system in female canaries

Songbirds have a specialized steroid‐sensitive network of brain nuclei, the song system, for controlling song. Most nuclei of the song system express androgen receptors, and the sensory‐motor integration nucleus High Vocal Center (HVC) alone also expresses estrogen receptors. Apart from expressing estrogen receptors in the vocal control system, songbirds are unique among birds because they have high concentrations of the estrogen‐synthesizing enzyme aromatase in the neostriatum surrounding HVC. However, the role of estrogen in controlling the development of the song structure has been scarcely investigated. In this work, we show that blocking the production of estrogen during testosterone‐induced song motor development in adult female canaries alters the song pattern compared to control females treated with testosterone only. These effects were correlated with inhibition of the expression of estrogen‐sensitive genes, such as brain‐derived nerve growth factor, in HVC. The expression of the ATP‐synthase gene, an indicator of cell activity, in HVC, and the size of HVC, were not affected by the treatment. Our results provide the first example of estrogen‐sensitive mechanisms controlling the structural features of adult birdsong. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 370–379, 2003     

Androgens and estrogens induce seasonal‐like growth of song nuclei in the adult songbird brain

In seasonally breeding songbirds, the brain regions that control song behavior undergo dramatic structural changes at the onset of each annual breeding season. As spring approaches and days get longer, gonadal testosterone (T) secretion increases and triggers the growth of several song control nuclei. T can be converted to androgenic and estrogenic metabolites by enzymes expressed in the brain. This opens the possibility that the effects of T may be mediated via the androgen receptor, the estrogen receptor, or both. To test this hypothesis, we examined the effects of two bioactive T metabolites on song nucleus growth and song behavior in adult male white‐crowned sparrows. Castrated sparrows with regressed song control nuclei were implanted with silastic capsules containing either crystalline T, 5α‐dihydrotestosterone (DHT), estradiol (E₂), or a combination of DHT+E₂. Control animals received empty implants. Song production was highly variable within treatment groups. Only one of seven birds treated with E₂ alone was observed singing, whereas a majority of birds with T or DHT sang. After 37 days of exposure to sex steroids, we measured the volumes of the forebrain song nucleus HVc, the robust nucleus of the archistriatum (RA), and a basal ganglia homolog (area X). All three steroid treatments increased the volumes of these three song nuclei when compared to blank‐implanted controls. These data demonstrate that androgen and estrogen receptor binding are sufficient to trigger seasonal song nucleus growth. These data also suggest that T's effects on seasonal song nucleus growth may depend, in part, upon enzymatic conversion of T to bioactive metabolites. © 2003 Wiley Periodicals, Inc. J Neurobiol 57:130–140, 2003     

Coordinated and dissociated effects of testosterone on singing behavior and song control nuclei in canaries (Serinus canaria)

Temperate zone songbirds that breed seasonally exhibit pronounced differences in reproductive behaviors including song inside and outside the breeding season. Springlike long daylengths are associated with increases in plasma testosterone (T) concentrations, as well as with increases in singing and in the volume of several brain nuclei known to control this behavior. The mechanisms whereby T can induce changes in behavior and brain, and whether or not these effects are differentially regulated, have recently begun to be examined, as has the question of the relative contributions of T and its androgenic and estrogenic metabolites to the regulation of this seasonal behavioral and neural plasticity. In this experiment, we examined the effects of T, 5alpha-dihydrotestosterone, or 17beta-estradiol treatment on castrated male canaries housed on short days and compared neural and behavioral effects in these males to similarly-housed males given only blank implants. We observed that only T treatment was effective in eliciting significant increases in singing behavior after 11 days of hormone exposure. In addition, T alone was effective in increasing the volume of a key song production nucleus, HVC. However, at this time, none of the steroids had any effects on the volumes of two other song control nuclei, Area X of the medial striatum and the robust nucleus of the arcopallium (RA), that are efferent targets of HVC, known to be regulated by androgen in canaries and also to play a role in the control of adult song. T can thus enhance singing well before concomitant androgen-induced changes in the song control system are complete.     

The song must go on: resilience of the songbird vocal motor pathway

Stereotyped sequences of neural activity underlie learned vocal behavior in songbirds; principle neurons in the cortical motor nucleus HVC fire in stereotyped sequences with millisecond precision across multiple renditions of a song. The geometry of neural connections underlying these sequences is not known in detail though feed-forward chains are commonly assumed in theoretical models of sequential neural activity. In songbirds, a well-defined cortical-thalamic motor circuit exists but little is known the fine-grain structure of connections within each song nucleus. To examine whether the structure of song is critically dependent on long-range connections within HVC, we bilaterally transected the nucleus along the anterior-posterior axis in normal-hearing and deafened birds. The disruption leads to a slowing of song as well as an increase in acoustic variability. These effects are reversed on a time-scale of days even in deafened birds or in birds that are prevented from singing post-transection. The stereotyped song of zebra finches includes acoustic details that span from milliseconds to seconds--one of the most precise learned behaviors in the animal kingdom. This detailed motor pattern is resilient to disruption of connections at the cortical level, and the details of song variability and duration are maintained by offline homeostasis of the song circuit.     

Androgen control of vocal control region volumes in a wild migratory songbird (Junco hyemalis) is region and possibly age dependent

Previous laboratory studies have shown that photoperiodic adult songbirds experience seasonal variations in singing frequency that correlate with plasma androgen levels, as well as changes in the brain regions that control singing (vocal control regions). The present study investigates naturally occurring seasonal changes in the sizes of these regions in a wild migratory species (dark-eyed junco, Junco hyemalis), with samples from adolescence to post-breeding fall migration. In adult males, the volumes of the vocal control regions area X and the higher vocal center (HVC) were large during the breeding season when birds were singing and androgen levels were high, and decreased in size after the breeding season when singing had stopped and androgen levels were low. HVC volume in adolescent males caught in the fall (no singing), when plasma androgen levels were low, was smaller than in breeding adults, thereby following the seasonal pattern of change in plasma androgen levels. In adolescent males, however, area X volume was the same as in breeding adults. Thus, area X size in adolescent male juncos may be testosterone independent. The seasonal pattern of robust nucleus of the archistriatum volume was similar to that of the HVC. The volumes of neither the magnocellular nucleus of the anterior neostriatum nor the nucleus rotundus, a control region, differed seasonally. Castration of breeding adult males caused both area X and HVC volumes to decrease compared to castrated controls with testosterone replacement, indicating that maintenance of these two region volumes is testosterone dependent in adults. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 391–402, 1997.     

Birth,migration, incorporation,and death of vocal control neurons in adult songbirds

Neurogenesis continues in the brain of adult birds. These cells are born in the ventricular zone of the lateral ventricles. Young neurons then migrate long distances guided, in part, by radial cell processes and become incorporated throughout most of the telencephalon. In songbirds, the high vocal center (HVC), which is important for the production of learned song, receives many of its neurons after hatching. HVC neurons which project to the robust nucleus of the archistriatum to form part of the efferent pathway for song production, and HVC interneurons continue to be added throughout life. In contrast, Area X-projecting HVC cells, thought to be part of a circuit necessary for song learning but not essential for adult song production, are only born in the embryo. New neurons in HVC of juvenile and adult birds replace older cells that die. There is a correlation between seasonal cell turnover rates (addition and loss) and testosterone levels in adult male canaries. Available evidence suggests that steroid hormones control the recruitment and/or survival of new HVC neurons, but not their production. The functions of neuronal replacement in adult birds remain unclear. However, rates of HVC neuron turnover are highest at times of year when canaries modify their songs. Replaceable HVC neurons may participate in the modification of perceptual memories or motor programs for song production. In contrast, permanent HVC neurons could hold long-lasting song-related information. The unexpected large-scale production of neurons in the adult brain holds important clues about brain function and, in particular, about the neural control of a learned behavior—birdsong. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 585–601, 1997     

雄激素调控鸣禽鸣唱核团对鸣唱行为影响

鸣禽的鸣唱是一种习得性行为,它由脑内离散的神经核团所控制,这些核团相互关联构成鸣唱控制系统.鸣禽体内的性激素可以通过调控鸣唱系统来影响鸣唱行为.研究表明性激素中的雄激素在调节鸣唱稳定性方面发挥关键作用.雄激素可以通过调控细胞增殖、神经元电生理特性、突触传递及相关受体来影响鸣唱控制核团进而导致鸣唱行为改变.本文主要集中在雄激素对鸣禽鸣唱行为调控作用的神经机制研究进展进行论述.     

Androgens and estrogens induce seasonal-like growth of song nuclei in the adult songbird brain

In seasonally breeding songbirds, the brain regions that control song behavior undergo dramatic structural changes at the onset of each annual breeding season. As spring approaches and days get longer, gonadal testosterone (T) secretion increases and triggers the growth of several song control nuclei. T can be converted to androgenic and estrogenic metabolites by enzymes expressed in the brain. This opens the possibility that the effects of T may be mediated via the androgen receptor, the estrogen receptor, or both. To test this hypothesis, we examined the effects of two bioactive T metabolites on song nucleus growth and song behavior in adult male white-crowned sparrows. Castrated sparrows with regressed song control nuclei were implanted with silastic capsules containing either crystalline T, 5alpha-dihydrotestosterone (DHT), estradiol (E(2)), or a combination of DHT+E(2). Control animals received empty implants. Song production was highly variable within treatment groups. Only one of seven birds treated with E(2) alone was observed singing, whereas a majority of birds with T or DHT sang. After 37 days of exposure to sex steroids, we measured the volumes of the forebrain song nucleus HVc, the robust nucleus of the archistriatum (RA), and a basal ganglia homolog (area X). All three steroid treatments increased the volumes of these three song nuclei when compared to blank-implanted controls. These data demonstrate that androgen and estrogen receptor binding are sufficient to trigger seasonal song nucleus growth. These data also suggest that T's effects on seasonal song nucleus growth may depend, in part, upon enzymatic conversion of T to bioactive metabolites.     

A neural network model for generating complex birdsong syntax

The singing behavior of songbirds has been investigated as a model of sequence learning and production. The song of the Bengalese finch, Lonchura striata var. domestica, is well described by a finite state automaton including a stochastic transition of the note sequence, which can be regarded as a higher-order Markov process. Focusing on the neural structure of songbirds, we propose a neural network model that generates higher-order Markov processes. The neurons in the robust nucleus of the archistriatum (RA) encode each note; they are activated by RA-projecting neurons in the HVC (used as a proper name). We hypothesize that the same note included in different chunks is encoded by distinct RA-projecting neuron groups. From this assumption, the output sequence of RA is a higher-order Markov process, even though the RA-projecting neurons in the HVC fire on first-order Markov processes. We developed a neural network model of the local circuits in the HVC that explains the mechanism by which RA-projecting neurons transit stochastically on first-order Markov processes. Numerical simulation showed that this model can generate first-order Markov process song sequences.     

Differential estrogen accumulation among populations of projection neurons in the higher vocal center of male canaries

The higher vocal center (HVC) of adult male canries undergoes a seasonal change in volume that corresponds to seasonal modifications of vocal behavior: HVC is large when birds produce stereotyped song (spring) and is small when birds produce plastic song and add new song syllables into their vocal repertoires (fall). We reported previously that systemic exposure to testosterone (T) produces an increase in the volume of HVC similar to that observed with long-day photoperiods. T-induced growth of HVC occured regardless of wheter the borders of HVC were defined by Nissl-staining, the distribution of androgen-concentrating cells, or the distribution of projection neurons [separate neuronal populations within HVC project to the robust nucleus of the archistriatum (RA) and to Area X of the avian striatum (X)]. In the present study we used steroid autoradiography to determine whether T can influence the distribution of HVC cells that bind estrogen, and we combined estrogen autoradiography with retrograde labeling to determine whether HVC neurons that project to RA versus X differ in their ability to accumulate estrogen. Results showed that T increased the volume of Nissl-defined HVC and although HVC contained a low density of estrogen-concentrating cells, T increased the spatial distribution of these cells to match the Nissl borders of HVC. We also identified a region containing a high density of estrogenconcentrating cells located medial to HVC [we call this region paraHVC (pHVC)], and T also increased the volume of pHVC. pHVC also contained numerous X-projecting neurons, but few if any RA-projecting neurons. Double-labeling analysis revealed the RA-projecting neurons did not accumulate estrogen, a small percentage of X-projecting neurons in HVC accumulated estrogen, and the majority of X-projecting neurons in pHVC showed heavy accumulation of estrogen. The data reported here and in our previous article suggest distinct roles for gonadal steroids within the HVC-pHVC complex: estrogens are concentrated by neurons that project to a striatal region that influences vocal production during song learning (X), whereas androgens are concentrated primarily by neurons that project to a motor region that is involved in vocal production during both song learning and the recitation of already-learned song (RA). © 1995 John Wiley & Sons, Inc.     

Immediate early gene activity in song control nuclei and brain areas regulating motivation relates positively to singing behavior during, but not outside of, a breeding context

In some species, such as songbirds, much is known about how the brain regulates vocal learning, production, and perception. What remains a mystery is what regulates the motivation to communicate. European starlings (Sturnus vulgaris) sing throughout most of the year, but the social and environmental factors that motivate singing behavior differ seasonally. Male song is highly sexually motivated during, but not outside of, the breeding season. Brain areas outside the song control system, such as the medial preoptic nucleus (POM) and ventral tegmental area (VTA), have been implicated in regulating sexually motivated behaviors in birds, including song. The present study was designed to explore whether these regions, as well as three song control nuclei [area X, the high vocal center (HVC), and the robust nucleus of the arcopallium (RA)], might be involved differentially in song produced within compared to outside of a breeding context. We recorded the behavioral responses of breeding and nonbreeding condition male starlings to the introduction of a female conspecific. Males did not show context-dependent differences in the overall amount of song sung. However, immunocytochemistry for the protein product of the immediate early gene cFOS revealed a positive linear relationship between the total amount of songs sung and number of cFOS-labeled cells in POM, VTA, HVC, and RA for birds singing during, but not outside of, a breeding context. These results suggest that these regions differentially regulate male song production depending on reproductive context. Overall the data support the hypothesis that the POM and VTA interact with the song control system, specifically HVC and RA, to regulate sexually motivated vocal communication in songbirds.     

Song environment affects singing effort and vasotocin immunoreactivity in the forebrain of male Lincoln's sparrows

Male songbirds often establish territories and attract mates by singing, and some song features can reflect the singer's condition or quality. The quality of the song environment can change, so male songbirds should benefit from assessing the competitiveness of the song environment and appropriately adjusting their own singing behavior and the neural substrates by which song is controlled. In a wide range of taxa, social modulation of behavior is partly mediated by the arginine vasopressin or vasotocin (AVP/AVT) systems. To examine the modulation of singing behavior in response to the quality of the song environment, we compared the song output of laboratory-housed male Lincoln's sparrows (Melospiza lincolnii) exposed to 1 week of chronic playback of songs categorized as either high or low quality, based on song length, complexity, and trill performance. To explore the neural basis of any facultative shifts in behavior, we also quantified the subjects' AVT immunoreactivity (AVT-IR) in three forebrain regions that regulate sociosexual behavior: the medial bed nucleus of the stria terminalis (BSTm), the lateral septum (LS), and the preoptic area. We found that high-quality songs increased singing effort and reduced AVT-IR in the BSTm and LS, relative to low-quality songs. The effect of the quality of the song environment on both singing effort and forebrain AVT-IR raises the hypothesis that AVT within these brain regions plays a role in the modulation of behavior in response to competition that individual males may assess from the prevailing song environment.     

Sex and seasonal differences in neurogenesis and volume of the song‐control system are associated with song in brood‐parasitic and non‐brood‐parasitic icterid songbirds

The song‐control system in the brain of songbirds is important for the production and acquisition of song and exhibits both remarkable seasonal plasticity and some of the largest neural sex differences observed in vertebrates. We measured sex and seasonal differences in two nuclei of the song‐control system of brood‐parasitic brown‐headed cowbirds (Molothrus ater) and closely‐related non‐parasitic red‐winged blackbirds (Agelaius phoeniceus). These species differ in both the development and function of song. Brown‐headed cowbirds have a larger sex difference in song than red‐winged blackbirds. Female cowbirds never sing, whereas female blackbirds do though much less than males. In cowbirds, song primarily functions in mate choice and males modify their song as they approach sexual maturity and interact with females. In red‐winged blackbirds, song is used primarily in territorial defence and is crystalized earlier in life. We found that the HVC was more likely to be discernable in breeding female blackbirds than in breeding female cowbirds. Compared to males, females had a smaller HVC and a smaller robust nucleus of the arcopallium (RA). However, females had higher doublecortin immunoreactivity (DCX+) in HVC, a measure of neurogenesis. Consistent with sex differences in song, the sex difference in RA volume was greater in cowbirds than in blackbirds. Males of both species had a smaller HVC with higher DCX+ in post‐breeding condition than in breeding condition when song is more plastic. Sex and seasonal differences in the song‐control system were closely related to variation in song in these two icterid songbirds. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1226–1240, 2016     

Social context affects testosterone-induced singing and the volume of song control nuclei in male canaries (Serinus canaria)

The contribution of social factors to seasonal plasticity in singing behavior and forebrain nuclei controlling song, and their interplay with gonadal steroid hormones are still poorly understood. In many songbird species, testosterone (T) enhances singing behavior but elevated plasma T concentrations are not absolutely required for singing to occur. Singing is generally produced either to defend a territory or to attract a mate and it is therefore not surprising that singing rate can be influenced by the sex and behavior of the social partner. We investigated, based on two independent experiments, the effect of the presence of a male or female partner on the rate of song produced by male canaries. In the first experiment, song rate was measured in dyads composed of one male and one female (M-F) or two males (M-M). Birds were implanted with T-filled Silastic capsules or with empty capsules as control. The number of complete song bouts produced by all males was recorded during 240 min on week 1, 2, 4, and 8 after implantation. On the day following each recording session, brains from approximately one-fourth of the birds were collected and the volumes of the song control nuclei HVC and RA were measured. T increased the singing rate and volume of HVC and RA but these effects were affected by the social context. Singing rates were higher in the M-M than in the M-F dyads. Also, in the M-M dyads a dominance-subordination relationship soon became established and dominant males sang at higher rates than subordinates in T-treated but not in control pairs. The differences in song production were not reflected in the size of the song control nuclei: HVC was larger in M-F than in M-M males and within the M-M dyads, no difference in HVC or RA size could be detected between dominant and subordinate males. At the individual level, the song rate with was positively correlated with RA and to a lower degree HVC volume, but this relationship was observed only in M-M dyads, specifically in dominant males. A second experiment, carried out with castrated males that were all treated with T and exposed either to another T-treated castrate or to an estradiol-implanted female, confirmed that song rate was higher in the M-M than in the M-F condition and that HVC volume was larger in heterosexual than in same-sex dyads. The effects of T on singing rate and on the volume of the song control nuclei are thus modulated by the social environment, including the presence/absence of a potential mate and dominance status among males.     

Testosterone regulates the activity and expression of aromatase in the canary neostriatum

The estrogen synthesizing enzyme, P450 aromatase, plays a critical role in the regulation of vertebrate sexual behavior. Songbirds differ from other avian species in the distribution and expression of aromatase in the telencephalon. The highest concentration of aromatase in the songbird brain is found in the caudomedial neostriatum (NCM). This area surrounds the only nucleus of the neural song system that contains estrogen receptors, the high vocal center (HVC). It has been suggested that estrogen produced in NCM via aromatization of circulating testosterone (T) is involved in song development and adult song plasticity. The modalities of regulation of aromatase in NCM are not well understood, and some studies suggest that in NCM, unlike in the preoptic-hypothalamic areas, aromatase is not regulated by androgen and/or estrogen. In this work, we studied whether the treatment of female canaries with T, which induces the development of malelike song and the masculinization of the song system, also induces an increase in the expression and activity of aromatase in NCM. Our results show that both the expression and activity of aromatase in NCM increase in female canaries following T treatment. This study provides the first direct evidence that T regulates telencephalic aromatase in songbirds, and suggests that an increase in estrogen production in NCM might be functional in neural and behavioral plasticity during phases of song organization.