Reward Associated with Singing Behavior Correlates with Opioid-Related Gene Expression in the Medial Preoptic Nucleus in Male European Starlings

Birdsong consists of species-specific learned vocal sequences that are used primarily to attract mates and to repel competitors during the breeding season. However, many birds continue to sing at times when vocal production has no immediate or obvious impact on conspecific behavior. The mechanisms that ensure that animals produce important behaviors in contexts in which the function of these behaviors is not immediate or obvious are not known. One possibility is that animals engage in such behaviors because they are associated with pleasure. Here we examined the hypothesis that male European starlings sing outside of the breeding season in part because the act of singing in this context is facilitated and/or maintained by opioid-mediated reward. We measured song-associated reward using a conditioned place preference (CPP) test in male starlings producing fall, non-breeding season-typical song. We used quantitative real time PCR to measure expression of the enkephalin opioid precursor preproenkephalin (PENK) and mu opioid receptors (MOR) in the medial preoptic nucleus (POM; a region in which opioids are implicated in both reward and starling fall song) and additionally the song control region HVC as a control. Starlings developed a strong preference for a place that had been paired previously with the act of producing fall-typical song, indicating that fall song production was associated with a positive affective state. Both PENK and MOR mRNA expression in the POM, but not HVC, correlated positively with both individual reward state (as reflected in CPP) and undirected singing behavior. These results suggest that singing induces opioid receptor and enkephalin expression in the POM and consequent reward, and/or that opioid release in the POM induced by individual or environmental factors (e.g., the presence of food, safety of a flock or the absence of predators) induces a positive affective state which then facilitates singing behavior.     

Social context-induced song variation affects female behavior and gene expression

Social cues modulate the performance of communicative behaviors in a range of species, including humans, and such changes can make the communication signal more salient. In songbirds, males use song to attract females, and song organization can differ depending on the audience to which a male sings. For example, male zebra finches (Taeniopygia guttata) change their songs in subtle ways when singing to a female (directed song) compared with when they sing in isolation (undirected song), and some of these changes depend on altered neural activity from a specialized forebrain-basal ganglia circuit, the anterior forebrain pathway (AFP). In particular, variable activity in the AFP during undirected song is thought to actively enable syllable variability, whereas the lower and less-variable AFP firing during directed singing is associated with more stereotyped song. Consequently, directed song has been suggested to reflect a “performance” state, and undirected song a form of vocal motor “exploration.” However, this hypothesis predicts that directed–undirected song differences, despite their subtlety, should matter to female zebra finches, which is a question that has not been investigated. We tested female preferences for this natural variation in song in a behavioral approach assay, and we found that both mated and socially naive females could discriminate between directed and undirected song—and strongly preferred directed song. These preferences, which appeared to reflect attention especially to aspects of song variability controlled by the AFP, were enhanced by experience, as they were strongest for mated females responding to their mate's directed songs. We then measured neural activity using expression of the immediate early gene product ZENK, and found that social context and song familiarity differentially modulated the number of ZENK-expressing cells in telencephalic auditory areas. Specifically, the number of ZENK-expressing cells in the caudomedial mesopallium (CMM) was most affected by whether a song was directed or undirected, whereas the caudomedial nidopallium (NCM) was most affected by whether a song was familiar or unfamiliar. Together these data demonstrate that females detect and prefer the features of directed song and suggest that high-level auditory areas including the CMM are involved in this social perception.     

Social Context–Induced Song Variation Affects Female Behavior and Gene Expression

Social cues modulate the performance of communicative behaviors in a range of species, including humans, and such changes can make the communication signal more salient. In songbirds, males use song to attract females, and song organization can differ depending on the audience to which a male sings. For example, male zebra finches (Taeniopygia guttata) change their songs in subtle ways when singing to a female (directed song) compared with when they sing in isolation (undirected song), and some of these changes depend on altered neural activity from a specialized forebrain-basal ganglia circuit, the anterior forebrain pathway (AFP). In particular, variable activity in the AFP during undirected song is thought to actively enable syllable variability, whereas the lower and less-variable AFP firing during directed singing is associated with more stereotyped song. Consequently, directed song has been suggested to reflect a “performance” state, and undirected song a form of vocal motor “exploration.” However, this hypothesis predicts that directed–undirected song differences, despite their subtlety, should matter to female zebra finches, which is a question that has not been investigated. We tested female preferences for this natural variation in song in a behavioral approach assay, and we found that both mated and socially naive females could discriminate between directed and undirected song—and strongly preferred directed song. These preferences, which appeared to reflect attention especially to aspects of song variability controlled by the AFP, were enhanced by experience, as they were strongest for mated females responding to their mate's directed songs. We then measured neural activity using expression of the immediate early gene product ZENK, and found that social context and song familiarity differentially modulated the number of ZENK-expressing cells in telencephalic auditory areas. Specifically, the number of ZENK-expressing cells in the caudomedial mesopallium (CMM) was most affected by whether a song was directed or undirected, whereas the caudomedial nidopallium (NCM) was most affected by whether a song was familiar or unfamiliar. Together these data demonstrate that females detect and prefer the features of directed song and suggest that high-level auditory areas including the CMM are involved in this social perception.     

Socially induced brain differentiation in a cooperatively breeding songbird

Birds living in social groups establish dominance hierarchies, and taking up the dominant position influences behaviour and physiological parameters. In cooperatively breeding white-browed sparrow weavers (Plocepasser mahali), the transition from subordinate helper to dominant breeder male induces the production of a new type of song. This song contains a large number of new syllables and differs in temporal pattern from duet songs produced by all other group members. Here we show that this change in social status of adult males affects the morphology of a behavioural control circuit, the song control system of songbirds that is composed of large neuron populations. The volume of the song control areas HVC and RA and their gene-expression levels depend on males' social status. Dominant males have several times larger testes than subordinates, which is not reflected in circulating androgen and oestrogen levels. Our findings suggest a remarkable differentiation of adult vertebrate brains in relation to changing social cues.     

Undirected Song Encourages the Breeding Female Zebra Finch to Remain in the Nest

Wild zebra finches sing frequently during the breeding season, but the vast majority of song is of the undirected song type that is not directed at any individual, and the function of which is obscure — it appears to be ignored by all potential recipients. It is sung close to the nest-site, has a peak in production during the egg-laying period, and diminishes thereafter. The incidence of undirected song is positively correlated with extra-pair courtship, a finding consistent with the hypothesis that it is a means of advertising availability for extra-pair matings. Typically, undirected song occurred outside the nest when the female was inside, and a positive relationship was found between the amount of singing given by the male during the 5-min interval immediately after the female entered the nest and the time she subsequently spent inside the nest. Keeping the partner inside the nest during her fertile period is an advantage to the male: it serves as a form of paternity protection against other males and it allows him opportunities to pursue his own extra-pair matings. Occupancy of the nest during laying is also a means of guarding against intraspecific brood parasitism, which was high at this colony.     

Responses of a sub-oscine bird during playback: effects of different song variants and breeding period

Vermilion flycatchers (Pyrocephalus rubinus) vary their song rate and song length across the breeding season. Males sing more and longer songs after nest construction than before. Here we explored the possibility that this variation is meaningful to territorial males. Using a playback approach, we tested several males with different variations in song output (i.e. variations in song length and song rate) in different periods of the breeding season (i.e. before and after the onset of nest construction). We found that males call more in response to playbacks of long and short songs before the onset of nest construction. However, after nest construction began they responded flying more when exposed to long songs than to short songs. These results show that vermilion flycatcher discriminates between different variants of song length, and suggest that males react to long songs as if they were more threatening signals than short songs, especially after the onset of nest construction. We did not find evidence of males discriminating between a high and a low song rate. We discuss some possible implications for song function in this sub-oscine species, and compare these findings with other results in oscine species.     

Assessing visual requirements for social context-dependent activation of the songbird song system

Social context has been shown to have a profound influence on brain activation in a wide range of vertebrate species. Best studied in songbirds, when males sing undirected song, the level of neural activity and expression of immediate early genes (IEGs) in several song nuclei is dramatically higher or lower than when they sing directed song to other birds, particularly females. This differential social context-dependent activation is independent of auditory input and is not simply dependent on the motor act of singing. These findings suggested that the critical sensory modality driving social context-dependent differences in the brain could be visual cues. Here, we tested this hypothesis by examining IEG activation in song nuclei in hemispheres to which visual input was normal or blocked. We found that covering one eye blocked visually induced IEG expression throughout both contralateral visual pathways of the brain, and reduced activation of the contralateral ventral tegmental area, a non-visual midbrain motivation-related area affected by social context. However, blocking visual input had no effect on the social context-dependent activation of the contralateral song nuclei during female-directed singing. Our findings suggest that individual sensory modalities are not direct driving forces for the social context differences in song nuclei during singing. Rather, these social context differences in brain activation appear to depend more on the general sense that another individual is present.     

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.     

The Onset of Song Learning and Song Tutor Selection in Fledgling Zebra Finches

We investigated song development in the pre‐independent zebra finch (aged 15–35 d), a period when neural pathways for song learning and production are forming and social influences outside the family are limited. Expt 1 investigated the onset and the minimum duration of tutoring needed for song learning in fledglings. We found most begin to learn song from 25 d of age and need about 10 d contact with the father tutor to make accurate copies. This onset corresponds with major developments in the formation of the neural circuitry implicated in song acquisition. Subsong also begins on day 25 suggesting that the sensory and motor phases of song learning fully overlap in the zebra finch. Our findings support the hypothesis that the song circuitry is fully functional by 35 d of age and the sensitive phase for zebra finches extends therefore from about days 25–65. However, only the first 10 d of this period are necessary to learn a tutor's song with fair accuracy. Expt 2 investigated the role of the paternal bond, spatial proximity and mating status in a fledgling's choice of song tutor where the father was the sole parent. Young chose the father over single unrelated males (expt 2a) or unrelated males in company with their female partners (expt 2b). Given the close spatial proximity of both potential tutors to the fledglings it is probably the filial bond, established via paternal care that is the cause of this preference. Zebra finches sing the same song phrase in two contrasting contexts: female‐directed song during pre‐copulatory courtship and undirected song where no female or display is involved. In expt 3 we tested the song learning preference of pre‐independent young for two categories of non‐paternal tutors: those singing predominantly female‐directed song and those singing exclusively undirected song. There was a small, but significant, preference for fledgling zebra finches to copy songs from males that sang female‐directed song. This preference is consistent with the hypothesis that young males not only learn the acoustic features of their tutor's song but also the visual and dynamic movements that constitute the courtship display.     

Minimal similarity in songs suggests limited exchange between humpback whales (Megaptera novaeangliae) in the southern Indian Ocean

Comparing humpback whale song from different breeding assemblages can reveal similarities in song due to acoustically interacting males, and therefore indirectly test whether males from different breeding sites are mixing. Northern Hemisphere song comparisons illustrated that whales within ocean basins share similar songs and are subpopulations within a larger population, whereas whales in different ocean basins are isolated populations and therefore do not share songs. During the 2006 breeding season, recordings were collected in Madagascar and Western Australia, and were compared visually plus aurally. Both regions shared one theme, whereas each region had four and six private themes, respectively. This study had a substantially low number of shared themes. The co‐occurrence of one theme was interpreted as an indication of limited exchange between these breeding assemblages, and we speculate that limited song similarity is due to inter‐oceanic interactions. Male(s) from an Indian Ocean breeding group could be exposed to novel song when they geographically overlap, and acoustically interact, with males from a different ocean basin. Novel song could induce rapid temporal changes as new song content is incorporated, thereby minimizing song similarities between that breeding group and other Indian Ocean breeding groups that were not exposed to the novel song.     

Facultative development of courtship and communication in juvenile male cowbirds (Molothrus ater)

We investigated effects of naturally occurring variation inexperience with adult males on development of song and courtshipcompetence in captive juvenile cowbirds. We studied birds ingroups housed in large outdoor aviaries that allowed birdsto regulate access to social stimulation. In two aviaries, we housed juvenile males and females either with or withoutadult males. Birds remained in these conditions from September1999 through their breeding season. We documented social andvocal development of juvenile males in the two aviaries bymeasuring social assortment and patterns and frequencies of their song interactions. We then brought the juveniles fromthe two aviaries together to compete against each other foraccess to females. In addition, we recorded juveniles' songsfour times over the study and played back their breeding seasonsongs to females in sound-attenuating chambers to measure the effectiveness of songs in eliciting copulatory responses fromthe females. Compared to juvenile males housed with adult males,juvenile males housed without adult males developed atypicalbehavior patterns. They (1) displayed little intrasexual aggressionor near-neighbor associations and (2) exhibited different patternsof courtship and copulation, but (3) were as successful at competing for copulations. Furthermore, they developed stereotypedsongs sooner and developed more potent breeding season songs.These different outcomes could not be traced to one variablebut to a cascade of effects involving diverging patterns ofsong acquisition and social interaction. The patterns of socialskills that emerged indicate considerable plasticity in the mechanisms underlying acquisition of courtship competence.     

Does age matter in song bird vocal interactions? Results from interactive playback experiments

The song of oscines provides an extensively studied model of age-dependent behaviour changes. Male and female receivers might use song characteristics to obtain information about the age of a signaller, which is often related to its quality. Whereas most of the age-dependent song changes have been studied in solo singing, the role of age in vocal interactions is less well understood. We addressed this issue in a playback study with common nightingales (Luscinia megarhynchos). Previous studies showed that male nightingales had smaller repertoires in their first year than older males and males adjusted their repertoire towards the most common songs in the breeding population. We now compared vocal interaction patterns in a playback study in 12 one year old and 12 older nightingales (cross-sectional approach). Five of these males were tested both in their first and second breeding season (longitudinal approach). Song duration and latency to respond did not differ between males of different ages in either approach. In the cross-sectional approach, one year old nightingales matched song types twice as often as did older birds. Similarly, in the longitudinal approach all except one bird reduced the number of song type matches in their second season. Individuals tended to overlap songs at higher rates in their second breeding season than in their first. The higher levels of song type matches in the first year and song overlapping by birds in their second year suggest that these are communicative strategies to establish relationships with competing males and/or choosy females.     

Validating the female bioassay of cowbird song: Relating differences in song potency to mating success

Female cowbirds (Molothrus ater), maintained in isolation from males during the breeding season, respond to the playback of male song with copulatory postures. They respond more often to some songs than others. Song potency can thus be operationally defined by the number of copulatory responses a song elicits. The purpose of the present study was to validate this measure of song potency by investigating its relationship to mating success. We observed two colonies of cowbirds during the breeding season and recorded details of their courtship. In addition, song potency of the males was tested by playback with a different group of captive females. The results indicate a relationship between maximum song potency and mating success: the males that obtained the most copulations had songs of higher maximum potency and were also observed to have been more dominant during the winter and early spring.     

Song, sexual selection, and a song control nucleus (HVc) in the brains of European sedge warblers

Female sedge warblers select males that have more complex songs as mates. This study tests two predictions concerning HVc, a telencephalic nucleus that is essential for song learning and production: first, that males with more complex songs will have a larger HVc, and second that males who pair successfully will have a larger HVc than unpaired males. Data on song composition and pairing status were collected from wild sedge warblers breeding in Hungary. We found significant positive correlations between three song attributes (repertoire size, song complexity, and song length) and the size of HVc. Males that paired successfully also had more complex songs (repertoire size and song complexity, though not song length) than males that did not. However, we find no direct evidence that males who paired successfully had a larger HVc than unpaired males. These findings are discussed in relation to the possible functions of HVc and also to current views on sexual selection and the evolution of the song control pathway.     

Ecological and Phylogenetic Effects on Song Sharing in Songbirds

As a consequence of vocal learning, the songs of male songbirds often vary geographically. We examined three proximate hypotheses that predict variation in the incidence of local song sharing among species: the annual cycle, breeding season duration, and epiphenomenon hypotheses. To test these hypotheses, we compiled data from the literature on local song sharing, annual cycle, breeding location isophane (an index of breeding season duration), repertoire size, and phylogenetic relationships of 65 populations in the family Fringillidae. Mapping characters on the phylogenetic tree revealed that local song sharing, migratory status, and breeding location isophane were randomly distributed on the phylogeny. Breeding isophane and migratory status were themselves correlated and had significant effects on the probability of sharing local songs. As predicted by the annual cycle and breeding season duration hypotheses, males in sedentary populations and populations breeding at low isophanes were more likely to share local songs. In subsets of the data, the annual cycle hypothesis received more support than did the breeding season duration hypothesis. Neither hypothesis explained more than 17% of the variation in the incidence of song sharing, and we suggest that detailed information on song function, male dispersal, and constraints on the timing of song learning will be needed to better predict the occurrence of local song sharing.     

Seasonality in song behaviour revisited: seasonal and annual variants and invariants in the song of the domesticated canary (Serinus canaria)

The song of the domesticated canary (Serinus canaria) is one of the most widely used models to study the neural correlates of behavioural plasticity and the mechanisms of female mate choice. However, only few studies have described the song behaviour in detail and monitored their changes throughout the year, and these data are restricted to the “Waterslager” strain. Here, we studied the song characteristics of the male common domesticated canary at different times of the year, the spring breeding and autumnal non-breeding season, and monitored the birds' songs up to the following breeding season. During breeding, males have increased plasma levels of testosterone, and songs are on average longer and consist of fewer non-repeated syllable types compared to the non-breeding season. When subsequent seasons are compared, song duration and the proportion of non-repeated syllable types change seasonally but not across years. Repertoire size remains constant throughout seasons although syllable types are exchanged. Syllable carry-over is significantly higher from one breeding season to the next than between the breeding and non-breeding season. Further, the repertoire of the breeding season contains more potentially sexually attractive syllable types than that of the non-breeding season. These data show that overall song structure is retained throughout the year while seasonality occurs in the temporal pattern and in repertoire composition.     

Sexual differentiation and seasonal variation in response to conspecific and heterospecific acoustic signals

Interspecific territoriality is frequently reported between closely related species; however, few studies have demonstrated interspecific territoriality between distantly related species living in sympatry. We conducted playback experiments to investigate territorial behaviour in male and female White‐bellied Wrens (Uropsila leucogastra) in response to simulated conspecific and heterospecific intruders during the breeding and non‐breeding seasons. We explored whether heterospecific songs of the Happy Wren (Pheugopedius felix), a distantly related species and ecological competitor, elicited antagonistic responses from focal White‐bellied Wrens, and whether such responses differed between the sexes. We also examined whether male and female responses to conspecific and heterospecific rivals varied with season. We found that male White‐bellied Wrens always responded to conspecific song, and responded significantly more to heterospecific song compared to a control stimulus (Tropical Parula, Setophaga pitiayumi). In contrast, although female White‐bellied Wrens responded strongly to conspecific song, their response to heterospecific song did not differ significantly from the control stimulus. The proportion of males that responded to heterospecific songs and the proportion of females that responded to conspecific songs varied seasonally, showing significantly lower responses during the breeding season. The intense responses of male White‐bellied Wrens to playback of heterospecific songs suggest that they recognise ecological competitors based on their vocal signals. Furthermore, the decrease in agonistic interactions during the breeding season is in line with the hypothesis that aggressive behaviour may be detrimental to reproductive and parental activity, and the hypothesis that heterospecific animals pose less of a threat during the breeding season.     

Male song diversity and its relation to breeding success in southern house wrens Troglodytes aedon chilensis

Theory proposes an adaptive relationship between male song complexity, including large song repertoires, and improved breeding success. Evidence supporting these relationships exists but is sometimes mixed or weak. Here we provide a first comprehensive study of the relationship between male song diversity and breeding success in a non‐migratory, austral population of house wrens Troglodytes aedon chilensis breeding in Mendoza, Argentina. During a two‐year field study, we measured breeding success for a population of 62 males and recorded more than 34 000 songs from a subsample of 26 males. For the latter subsample, we tested for correlations between six measures of song diversity and four canonical measures of annual breeding success. Males that sang with greater overall syllable type diversity and that had larger song repertories paired with females that bred earlier and laid more eggs over the course of the breeding season. However, these males also showed lower levels of immediate song type diversity, as measured by the Levenshtein distance between successive songs. We discuss implications for the evolution of song complexity in this exceptionally widespread species and the selective mechanisms that might influence song complexity in resident populations in the Neotropics compared to migratory populations in the northern hemisphere.     

First-Year Common Nightingales (Luscinia megarhynchos) Have Smaller Song-Type Repertoire Sizes Than Older Males

Based on the assumptions that birdsong indicates male quality and that quality is related to age, one might expect older birds to signal their age. That is, in addition to actual body condition, at least some song features should vary with age, presumably towards more complexity. We investigated this issue by comparing repertoire sizes of free‐ranging common nightingale males in their first breeding season with those of older males. Nightingales are a good model species as they are open‐ended learners, where song acquisition is not confined to an early sensitive period of learning. Moreover, nightingales develop an extraordinarily large song‐type repertoire (approx. 180 different song types per male), and differences in repertoire size among males are pronounced. We analysed repertoire characteristics of the nocturnal song of nine nightingales in their first breeding season and compared them with the songs of nine older males. The repertoire size of older males was on average 53% larger than that of yearlings. When analysing two song categories of nightingales, whistle and non‐whistle songs separately, we found similar results. Our findings show marked differences in repertoire size between age categories, suggesting that this song feature may reflect a male's age. We discuss those mechanisms that may constrain the development of larger repertoires in first‐year males. Whether repertoire sizes are crucial for female mate choice or in vocal interactions among conspecific males remains open to further investigations.     

Female dark‐eyed juncos Junco hyemalis thurberi produce male‐like song in a territorial context during the early breeding season

Reports of female song, once considered a rarity, have recently increased across a variety of avian taxa. Females of many species can be induced to produce male‐like song with exogenous testosterone, but observations of female song in free‐living birds remain limited by incomplete sampling of females. Here, we report three independent observations of female dark‐eyed juncos Junco hyemalis producing male‐like song early in the breeding season (i.e. post‐territory establishment, pre‐nesting) in a recently established non‐migratory, urban population. To elicit song, we presented 17 free‐living junco pairs with a live, caged female conspecific. Three unique females responded to our trials by diving at the intruding female, chasing their (male) mate, fanning their tail feathers, and singing a trilled song similar in structure to male long‐range (broadcast) song. We compared male and female songs quantitatively and found that the two sexes were statistically similar in many spectral and temporal characteristics, but female songs had significantly lower minimum and peak frequencies than males. This result is particularly surprising, as males in this urban population are known to sing at a significantly higher minimum frequency than males in a nearby montane population. Both the seasonal and social context in which these songs were observed suggest a potential function for female song in mate guarding and polygyny prevention, but more data are needed to test this hypothesis. Whether female song is common in all dark‐eyed juncos during the early breeding season or if it is restricted to this particular urban and non‐migratory population remains an important question for future research.