Stimulatory effects on the reproductive axis in female songbirds by conspecific and heterospecific male song

Courtship vocalizations of male songbirds can profoundly enhance the reproductive physiology and behavior of conspecific females. However, no study has fully investigated the selectivity of conspecific song effects on reproductive development in birds. We studied the effects of conspecific and heterospecific song on reproductive development in domesticated (canaries) and wild songbirds (song sparrows). As expected, conspecific song enhanced follicular development. Unexpectedly, however, birds exposed to heterospecific song also underwent enhanced follicular development (compared to birds exposed to no song); conspecific and heterospecific songs were equally effective in enhancing ovarian development. In canaries exposed to 18L:6D, conspecific song induced oviposition earlier and at a greater frequency than in heterospecific and no song groups, with the fewest eggs being laid in the no song group. These results indicate that conspecific and heterospecific male song can enhance reproductive activity in female songbirds. Whether or not activation of the reproductive axis in female songbirds by heterospecific song occurs in the wild remains unclear. It is also unclear as to whether the ability of the reproductive axis to respond to heterospecific song performs a specific function, or whether it is simply a consequence of greater selection pressure acting upon behavioral responses to song.     

Testosterone-induced changes in adult canary brain are reversible

Brain nuclei that control song are larger in male canaries, which sing, than in females, which sing rarely or not at all. Treatment of adult female canaries with testosterone (T) induces song production and causes song-control nuclei to grow, approaching the volumes observed in males. For example, the higher vocal center (HVC) of adult females approximately doubles in size by 1 month following the onset of T treatment. Male HVC projects to a second telencephalic nucleus, RA (the robust nucleus of the archistriatum), which projects in turn to the vocal motor neurons. Whether HVC makes a similar connection in female canaries is not known, although HVC and RA are not functionally connected in female zebra finches, a species in which testosterone does not induce neural or behavioral changes in the adult song system. This experiment investigated whether HVC makes an efferent projection to RA in normal adult female canaries, or if T is necessary to induce the growth of this connection. In addition, we examined whether T-induced changes in adult female canary brain are reversible. Adult female canaries received systemic T implants that were removed after 4 weeks; these birds were killed 4 weeks after T removal (Testosterone-Removal, T-R). Separate groups of control birds received either (a) T implants for 4 weeks which were not removed (Testosterone-Control, T-C) or (b) empty implants (Untreated Control, øO-C). Crystals of the fluorescent tracer DiI were placed in the song-control nucleus HVC in order to anterogradely label both efferent targets of HVC, RA and Area X. Projections from HVC to RA and Area X were present in all treatment groups including untreated controls, and did not appear to differ either qualitatively or quantitatively. Thus, formation of efferent connections from HVC may be prerequisite to hormone-induced expression of song behavior in adult songbirds. The volumes of RA and Area X were measured using the distribution of anterograde label as well as their appearance in Nissl-stained tissue. RA was larger in T-treated control birds than in untreated controls. Experimental birds in which T was given and then removed (T-R) had RA volumes closer in size to untreated controls (ø-C). Because the volume of RA in T-treated controls (T-C) was larger than that of birds that did not receive T (ø-C), we conclude that the volume of RA increased in both T-C and T-R birds but regressed upon removal of T in T-R birds. Surprisingly, the volume of Area X did not increase in T-treated birds. Birds in this study were maintained on short days, suggesting that T-induced growth of Area X reported previously may have resulted from an interaction between T and another seasonal or photoperiodic factor induced by exposure to long daylengths. © 1993 John Wiley & Sons, Inc.     

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     

HVC lesions modify immediate early gene expression in auditory forebrain regions of femalesongbirds

It is well established that auditory forebrain regions of oscine birds are essential for the encoding of species‐typical songs and are, therefore, vital for recognition of song during sociosexual interactions. Regions such as the caudal medial nidopallium (NCM) and the caudal medial mesopallium (CMM) are involved in perceptual processing of song and the formation of auditory memories. There is an additional telencephalic nucleus, however, that has also been implicated in species recognition. This nucleus is HVC, a prominent nucleus that sits at the apex of the song system, and is well known for its critical role in song learning and song production in male songbirds. Here, we explore the functional relationship between auditory forebrain regions (i.e., NCM and CMM) and HVC in female canaries (Serinus canaria). We lesion HVC and examine immediate early gene responses to conspecific song presentation within CMM and NCM to explore whether HVC can modulate auditory responses within these forebrain regions. Our results reveal robust deficits in ZENK‐ir in CMM and NCM of HVC‐lesioned females when compared with control‐ and sham‐lesioned females, indicating that functional connections exists between HVC and NCM/CMM. Although these connected regions have been implicated in song learning and production in males, they likely serve distinct functions in female songbirds that face the task of song recognition rather than song production. Identifying functional connections between HVC and auditory regions involved in song perception is an essential step toward developing a comprehensive understanding of the neural basis of song recognition. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Song learning in domesticated canaries in a restricted acoustic environment

Many songbirds learn their songs early in life from a song model. In the absence of such a model, they develop an improvised song that often lacks the species-typical song structure. Open-ended learners, such as the domesticated canary, are able to modify their songs in adulthood, although the mechanisms that guide and time the song-learning process are still not fully understood. In a previous study, we showed that male domesticated canaries lacking an adult song model in their first year substantially change their song repertoire and composition when exposed to normally reared conspecifics in their second year. Here, we investigate song development in descendants of canaries that were raised and kept as a peer group without a song model. Such males represent tutors with abnormal song characteristics. Interestingly, the F₁ generation developed quite normal song structure, and when brought into an environment with normally raised canaries in their second year, they did not modify their songs substantially. These results suggest that contact with an adult song model early in life is crucial for song crystallization, but also that song development is at least partly guided by innate rules. They also question the existing classification of canaries as open-ended learners.     

Hormonal acceleration of song development illuminates motor control mechanism in canaries

In songbirds, the ontogeny of singing behavior shows strong parallels with human speech learning. As in humans, development of learned vocal behavior requires exposure to an acoustic model of species‐typical vocalizations, and, subsequently, a sensorimotor practice period after which the vocalization is produced in a stereotyped manner. This requires mastering motor instructions driving the vocal organ and the respiratory system. Recently, it was shown that, in the case of canaries (Serinus canaria), the diverse syllables, constituting the song, are generated with air sac pressure patterns with characteristic shapes, remarkably, those belonging to a very specific mathematical family. Here, we treated juvenile canaries with testosterone at the onset of the sensorimotor practice period. This hormone exposure accelerated the development of song into stereotyped adultlike song. After 20 days of testosterone treatment, subsyringeal air sac pressure patterns of song resembled those produced by adults, while those of untreated control birds of the same age did not. Detailed temporal structure and modulation patterns emerged rapidly with testosterone treatment, and all previously identified categories of adult song were observed. This research shows that the known effect of testosterone on the neural circuits gives rise to the stereotyped categories of respiratory motor gestures. Extensive practice of these motor patterns during the sensorimotor phase is not required for their expression. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 943–960, 2010     

Male and female rufous‐and‐white wrens do not match song types with same‐sex rivals during simulated territorial intrusions

In birds with song repertoires, song‐type matching occurs when an individual responds to another individual's song by producing the same song type. Song‐type matching has been described in multiple bird species and a growing body of evidence suggests that song‐type matching may serve as a conventional signal of aggression, particularly in male birds in the temperate zone. Few studies have investigated song‐type matching in tropical birds or female birds, in spite of the fact that avian biodiversity is highest in the tropics, that female song is widespread in the tropics, and that female song is the ancestral state among songbirds. In this study of rufous‐and‐white wrens Thryophilus rufalbus, a resident neotropical songbird where both sexes sing, we presented territorial males and females with playback that simulated a territorial rival producing shared and unshared songs. In response, both males and females sang matched song types at levels statistically equal to levels expected by chance. Furthermore, males and females exhibited similar levels of aggression and similar vocal behaviours in response to playback of both shared and unshared songs. These results indicate that rufous‐and‐white wrens do not use song‐type matching in territorial conflicts as a conventional signal of aggression. We discuss alternative hypotheses for the function of song‐type sharing in tropical birds. In particular, we point out that shared songs may play an important role in intra‐pair communication, especially for birds where males and females combine their songs in vocal duets, and this may supersede the function of song‐type matching in some tropical birds.     

The effect of auditory distractors on song discrimination in male canaries (Serinus canaria)

Male songbirds such as canaries produce complex learned vocalizations that are used in the context of mate attraction and territory defense. Successful mate attraction or territorial defense requires that a bird be able to recognize individuals based on their vocal performance and identify these songs in a noisy background. In order to learn more about how birds are able to solve this problem, we investigated, with a two-alternative choice procedure, the ability of adult male canaries to discriminate between conspecific song segments from two different birds and to maintain this discrimination when conspecific songs are superimposed with a variety of distractors. The results indicate that male canaries have the ability to discriminate, with a high level of accuracy song segments produced by two different conspecific birds. Song discrimination was partially maintained when the stimuli were masked by auditory distractors, but the accuracy of the discrimination progressively declined as a function of the number of masking distractors. The type of distractor used in the experiments (other conspecific songs or different types of artificial white noise) did not markedly affect the rate of deterioration of the song discrimination. These data indicate that adult male canaries have the perceptual abilities to discriminate and selectively attend to one ongoing sound that occurs simultaneously with one or more other sounds. The administration of a noradrenergic neurotoxin did not impair markedly the discrimination learning abilities although the number of subjects tested was too small to allow any firm conclusion. In these conditions, however, the noradrenergic lesion significantly increased the number failures to respond in the discrimination learning task suggesting a role, in canaries, of the noradrenergic system in some attentional processes underlying song learning and processing.     

Auditory feedback is necessary for long-term maintenance of high-frequency sound syllables in the song of adult male budgerigars (<Emphasis Type="Italic">Melopsittacus undulatus</Emphasis>)

Among avian species that communicate using vocalization, songbirds (oscine Passeriformes), hummingbirds (Trochiliformes), and parrots (Psittaciformes) are vocal learners. Early studies showed that songbirds require auditory feedback for song development in young and maintenance in adults. To determine whether auditory feedback is also necessary for adult song maintenance in non-passerine species, we deprived adult male budgerigars (Psittaciformes) of auditory input by surgical cochlear removal. Songs of the deafened birds changed within 6 months after auditory deprivation. In postoperative songs, high narrowband syllables, which comprised frequency-modulated narrowband elements with relatively high fundamental frequencies of 2–4 kHz, decreased significantly. High harmonic broadband syllables, with fundamental frequencies ≥2 kHz, also decreased. The altered proportions of syllables were subsequently retained, and maintained 12 months after deafening. The sequence linearity score, a parameter representing the stereotypy of the syllable sequence, was higher than that before deafening. The inter-syllable silence was prolonged. Little change was observed in the songs of intact and sham-operated birds. The significant decrease in high-frequency syllables and song alteration followed by stabilization resembled the results with songbirds, although song stabilization took a long time in budgerigars. Therefore, our results suggest that psittacine budgerigars and oscine songbirds require auditory feedback similarly for adult song maintenance.     

Early life conditions that impact song learning in male zebra finches also impact neural and behavioral responses to song in females

Early life stressors can impair song in songbirds by negatively impacting brain development and subsequent learning. Even in species in which only males sing, early life stressors might also impact female behavior and its underlying neural mechanisms, but fewer studies have examined this possibility. We manipulated brood size in zebra finches to simultaneously examine the effects of developmental stress on male song learning and female behavioral and neural response to song. Although adult male HVC volume was unaffected, we found that males from larger broods imitated tutor song less accurately. In females, early condition did not affect the direction of song preference: all females preferred tutor song over unfamiliar song in an operant test. However, treatment did affect the magnitude of behavioral response to song: females from larger broods responded less during song preference trials. This difference in activity level did not reflect boldness per se, as a separate measure of this trait did not differ with brood size. Additionally, in females we found a treatment effect on expression of the immediate early gene ZENK in response to tutor song in brain regions involved in song perception (dNCM) and social motivation (LSc.vl, BSTm, TnA), but not in a region implicated in song memory (CMM). These results are consistent with the hypothesis that developmental stressors that impair song learning in male zebra finches also influence perceptual and/or motivational processes in females. However, our results suggest that the learning of tutor song by females is robust to disturbance by developmental stress. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018     

A field study of seasonal neuronal incorporation into the song control system of a songbird that lacks adult song learning

Adult songbirds can incorporate new neurons into HVc, a telencephalic song control nucleus. Neuronal incorporation into HVc is greater in the fall than in the spring in adult canaries (open‐ended song learners) and is temporally related to seasonal song modification. We used the western song sparrow, a species that does not modify its adult song, to test the hypothesis that neuronal incorporation into adult HVc is not seasonally variable in age‐limited song learners. Wild song sparrows were captured during the fall and the spring, implanted with osmotic pumps containing [³H]thymidine, released onto their territories, and recaptured after 30 days. The density, proportion, and number of new HVc neurons were all significantly greater in the fall than in the spring. There was also a seasonal change in the incorporation of new neurons into the adjacent neostriatum that was less pronounced than the change in HVc. This is the first study of neuronal recruitment into the song control system of freely ranging wild songbirds. These results indicate that seasonal changes in HVc neuronal incorporation are not restricted to open‐ended song learners. The functional significance of neuronal recruitment into HVc therefore remains elusive. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 316–326, 1999     

Hatching late in the season requires flexibility in the timing of song learning

Most songbirds learn their songs from adult tutors, who can be their father or other male conspecifics. However, the variables that control song learning in a natural social context are largely unknown. We investigated whether the time of hatching of male domesticated canaries has an impact on their song development and on the neuroendocrine parameters of the song control system. Average age difference between early- and late-hatched males was 50 days with a maximum of 90 days. Song activity of adult tutor males decreased significantly during the breeding season. While early-hatched males were exposed to tutor songs for on average the first 99 days, late-hatched peers heard adult song only during the first 48 days of life. Remarkably, although hatching late in the season negatively affected body condition, no differences between both groups of males were found in song characteristics either in autumn or in the following spring. Similarly, hatching date had no effect on song nucleus size and circulating testosterone levels. Our data suggest that late-hatched males must have undergone accelerated song development. Furthermore, the limited tutor song exposure did not affect adult song organization and song performance.     

Yolk testosterone, postnatal growth and song in male canaries

Avian eggs contain substantial amounts of maternal yolk androgens, which have been shown to modulate offspring phenotype. The first studies on the functional consequences of maternal yolk androgens have focused on early life stages and their role in sibling competition. However, recent longitudinal studies reported long-lasting effects of maternal yolk androgens on offspring phenotype, mostly concerning traits that are sensitive to androgens. This suggests that maternal yolk androgens could play an important role in sexual selection, since the expression of many male sexual characters is testosterone-dependent. Using male canaries as a model, we examined the consequences of an experimental elevation of yolk testosterone concentrations on early development as well as long-lasting effects particularly on song, which is one of the most important sexual characters in male songbirds. Elevated yolk testosterone concentrations inhibited male growth, possibly in interaction with an existent ectoparasite exposure. Males hatched from testosterone-treated eggs (T-males) did not have enhanced competitive skills, in contrast to previous studies. The elevation of yolk testosterone concentrations delayed song development but did not affect adult song phenotype. This is intriguing, as yolk testosterone possibly induced developmental stress, which is known to reduce song quality. We hypothesize that yolk testosterone has either no direct effect on adult song phenotype, or that positive effects are merged by the negative effects of developmental stress. Finally, females mated with T-males invested more in their clutch indicating that females either assess T-males as more attractive (differential allocation hypothesis) or compensated for lower offspring viability (compensation hypothesis).     

Changes in adult zebra finch song require a forebrain nucleus that is not necessary for song production

Male zebra finches normally crystallize song at approximately 90 days and do not show vocal plasticity as adults. However, changes to adult song do occur after unilateral tracheosyringeal (ts) nerve injury, which denervates one side of the vocal organ. We examined the effect of placing bilateral lesions in LMAN (a nucleus required for song development but not for song maintenance in adults) upon the song plasticity that is induced by ts nerve injury in adults. The songs of birds that received bilateral lesions within LMAN followed by right ts nerve injury silenced, on average, 0.25 syllables, and added 0.125 syllables (for an average turnover of 0.375 syllables), and changed neither the frequency with which individual syllables occurred within songs nor the motif types they used most often. In contrast, the songs of birds that received sham lesions followed by ts nerve injury lost, on average, 1.625 syllables, silenced 0.125 syllables, and added 0.75 syllables, turning over an average of 2.5 syllables. They also significantly changed both the frequency with which individual syllables were included in songs and the motif variants used. Thus, song plasticity induced in adult zebra finches with crystallized songs requires the presence of LMAN, a nucleus which had been thought to play a role in vocal production only during song learning. Although the changes to adult songs induced by nerve transection are more limited than those that arise during song development, the same circuitry appears to underlie both types of plasticity.     

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. 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Effects of Song Experience and Song Quality on Immediate Early Gene Expression in Female Canaries (Serinus canaria)

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.     

Embryonic exposure to environmentally relevant concentrations of a brominated flame retardant reduces the size of song‐control nuclei in a songbird

Environmental contaminants have the potential to act as developmental stressors and impair development of song and the brain of songbirds, but they have been largely unstudied in this context. 2,2′,4,4′,5‐Pentabromodiphenyl ether (BDE‐99) is a brominated flame retardant congener that has demonstrated endocrine disrupting effects, and has pervaded the global environment. We assessed the effects of in ovo exposure to environmentally relevant levels of BDE‐99 on the neuroanatomy of the song‐control system in a model songbird species, the zebra finch (Taeniopygia guttata). Embryos were exposed via egg injection to a vehicle control (DMSO), 10, 100, or 1000 ng BDE‐99/g egg on the day the egg was laid. Chicks were raised to sexual maturity to investigate long‐term effects of BDE‐99 on the adult male brain. Three key song‐control nuclei (Area X, HVC, RA) all showed a dose‐dependent trend toward decreasing volume as BDE‐99 concentration increased, and birds exposed to 1000 ng/g in ovo BDE‐99 had significantly smaller song‐control nuclei volume compared to control birds. High environmental concentrations of BDE‐99 in avian tissues can be within that range and thus could affect development of the song‐control system in birds, and potentially other processes. We previously found that BDE‐99 exposure during the nestling period had no effect of on the song‐control system, although it did have significant effects on some behaviural endpoints. Taken together, these results suggest that exposure to polybrominated diphenyl ether (PBDEs) during critical developmental windows can significantly alter neurological development. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018     

LMAN lesions prevent song degradation after deafening without reducing HVC neuron addition

In some songbirds perturbing auditory feedback can promote changes in song structure well beyond the end of song learning. One factor that may drive vocal change in such deafened birds is the ongoing addition of new vocal-motor neurons into the song system. Without auditory feedback to guide their incorporation, the addition of these new neurons could disrupt the established song pattern. To assess this hypothesis, the authors determined if neuronal recruitment into the vocal motor nucleus HVC is affected by neural signals that influence vocal change in adult deafened birds. Such signals appear to be conveyed via LMAN, a nucleus in the anterior forebrain that is necessary for vocal change after deafening. Here the authors tested whether LMAN lesions might restrict song degradation after deafening by reducing the addition or survival of new HVC neurons that would otherwise corrupt the ongoing song pattern. Using [3H]thymidine autoradiography to identify neurons generated in adult zebra finches, it was shown here that LMAN lesions do not reduce the number or percent of new HVC neurons surviving for either several weeks or months after [3H]thymidine labeling. However, the authors confirmed previous reports that LMAN lesions restrict vocal change after deafening. These data suggest that neurons incorporated into the adult HVC may form behaviorally adaptive connections without requiring auditory feedback, and that any role such neurons may play in promoting vocal change after adult deafening requires anterior forebrain pathway output.