Parasites affect song complexity and neural development in a songbird

There is now considerable evidence that female choice drives the evolution of song complexity in many songbird species. However, the underlying basis for such choice remains controversial. The developmental stress hypothesis suggests that early developmental conditions can mediate adult song complexity by perturbing investment in the underlying brain nuclei during their initial growth. Here, we show that adult male canaries (Serinus canaria), infected with malaria (Plasmodium relictum) as juveniles, develop simpler songs as adults compared to uninfected individuals, and exhibit reduced development of the high vocal centre (HVC) song nucleus in the brain. Our results show how developmental stress not only affects the expression of a sexually selected male trait, but also the structure of the underlying song control pathway in the brain, providing a direct link between brain and behaviour. This novel experimental evidence tests both proximate and ultimate reasons for the evolution of complex songs and supports the Hamilton-Zuk hypothesis of parasite-mediated sexual selection. Together, these results propose how developmental costs may help to explain the evolution of honest advertising in the complex songs of birds.     

Variation in the volume of zebra finch song control nuclei is heritable: developmental and evolutionary implications.

In many songbird species, females prefer males that sing a larger repertoire of syllables. Males with more elaborate songs have a larger high vocal centre (HVC) nucleus, the highest structure in the song production pathway. HVC size is thus a potential target of sexual selection. Here we provide evidence that the size of the HVC and other song production nuclei are heritable across individual males within a species. In contrast, we find that heritabilities of other nuclei in a song-learning pathway are lower, suggesting that variation in the sizes of these structures is more closely tied to developmental and environmental differences between individuals. We find that evolvability, a statistical measure that predicts response to selection, is higher for the HVC and its target for song production, the robustus archistriatalis (RA), than for all other brain volumes measured. This suggests that selection based on the functions of these two structures would result in rapid major shifts in their anatomy. We also show that the size of each song control nucleus is significantly correlated with the song related nuclei to which it is monosynaptically connected. Finally, we find that the volume of the telencephalon is larger in males than in females. These findings begin to join theoretical analyses of the role of female choice in the evolution of bird song to neurobiological mechanisms by which the evolutionary changes in behaviour are expressed.     

Female canaries that respond and discriminate more between male songs of different quality have a larger song control nucleus (HVC) in the brain

In male songbirds the song control pathway in the forebrain is responsible for song production and learning. In most species, females do not sing and have smaller nuclei in the song control pathway. Although the function of the pathway in females is assumed to be associated with the perception of male song, there is little direct evidence to support this view. In this study on female canaries, we investigate the role of two key nuclei in the song control pathway (HVC and lMAN) in relation to playback of male song. Male canaries produce elaborate songs that function to attract and stimulate females. The songs are constructed from smaller units called syllables, and special syllables with a more complex structure (sexy syllables) are known to induce females to perform copulation solicitation displays (CSD) as an invitation to mate. By using computer-edited experimental songs, we first show that females discriminate between songs by producing significantly more CSD to those containing sexy syllables. We then sectioned the brains and used in situ hybridization to reveal song nuclei containing androgen receptors. We report positive correlations between the size of HVC and both total CSD response and the amount of discrimination between sexy and nonsexy songs. We found no such relationships between these measures and the size of lMAN. These results provide some evidence to support the view that, in female canaries HVC is involved in female perception and discrimination of male song. The results also have implications for the evolution of complex male songs by sexual selection and female choice.     

Song as an honest signal of developmental stress in the zebra finch (Taeniopygia guttata)

In a wide range of bird species, females have been shown to express active preferences for males that sing more complex songs. Current sexual selection theory predicts that for this signal to remain an honest indicator of male quality, it must be associated with an underlying cost of development or maintenance. There has been considerable debate questioning the costs associated with song production and learning. Recently, the nutritional stress hypothesis proposed that song complexity could act as an indicator of early developmental history, since the song control nuclei in the brain are laid down early in life. Here we test the nutritional stress hypothesis, by investigating the effects of dietary stress on the quality of adult song produced. In addition, we tested the effects of elevated corticosterone during development on song production to test its possible involvement in mediating the effects of developmental stress. The results demonstrate that both dietary restriction and elevated corticosterone levels significantly reduced nestling growth rates. In addition, we found that experimentally stressed birds developed songs with significantly shorter song motif duration and reduced complexity. These results provide novel experimental evidence that complex song repertoires may have evolved as honest signals of male quality, by indicating early developmental rearing conditions.     

Early condition, song learning, and the volume of song brain nuclei in the zebra finch (Taeniopygia guttata)

Songbirds are an important model system for the study of the neurological bases of song learning, but variation in song learning accuracy and adult song complexity remains poorly understood. Current models of sexual selection predict that signals such as song must be costly to develop or maintain to constitute honest indicators of male quality. It has been proposed that reductions of nestling condition during song development might limit the expression of song learning. Adult song could thus act as an indicator of early stress as only males that enjoy good condition during development could learn accurately and sing long songs or large repertoires. We tested this hypothesis in the zebra finch by modifying early condition through cross-fostering chicks to small, medium, and large broods. Song learning was very accurate and was found to reflect very closely tutor song characteristics and to depend on the number of males in the tutoring group. Although the brood size manipulation strongly affected several measures of nestling condition and adult biometry, we found no relationship between early condition and song learning scores or song characteristics. Similarly, brain mass and high vocal center (HVC), robust nucleus of the arcopallium (RA), and lateral magnocellular nucleus of the anterior nidopallium (LMAN) volumes did not covary with nestling condition and growth measurements. We found no significant relationship between song repertoire size and HVC and RA volumes, although there was a nonsignificant trend for HVC to increase with increasing proportion of learnt elements in a song. In conclusion, the results provide no evidence for song learning to be limited by nestling condition during the period of nutritional dependence from the parents in this species.     

ENVIRONMENTAL AND GENETIC CONTROL OF BRAIN AND SONG STRUCTURE IN THE ZEBRA FINCH

Birdsong is a classic example of a learned trait with cultural inheritance, with selection acting on trait expression. To understand how song responds to selection, it is vital to determine the extent to which variation in song learning and neuroanatomy is attributable to genetic variation, environmental conditions, or their interactions. Using a partial cross fostering design with an experimental stressor, we quantified the heritability of song structure and key brain nuclei in the song control system of the zebra finch and the genotype‐by‐environment (G × E) interactions. Neuroanatomy and song structure both showed low levels of heritability and are unlikely to be under selection as indicators of genetic quality. HVC, in particular, was almost entirely under environmental control. G × E interaction was important for brain development and may provide a mechanism by which additive genetic variation is maintained, which in turn may promote sexual selection through female choice. Our study suggests that selection may act on the genes determining vocal learning, rather than directly on the underlying neuroanatomy, and emphasizes the fundamental importance of environmental conditions for vocal learning and neural development in songbirds.     

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.     

Early nutritional stress impairs development of a song-control brain region in both male and female juvenile song sparrows (Melospiza melodia) at the onset of song learning

Birdsong is a sexually selected trait and is often viewed as an indicator of male quality. The developmental stress hypothesis proposes a model by which song could be an indicator; the time during early development, when birds learn complex songs and/or local variants of song, is of rapid development and nutritional stress. Birds that cope best with this stress may better learn to produce the most effective songs. The developmental stress hypothesis predicts that early food restriction should impair development of song-control brain regions at the onset of song learning. We examined the effect of food restriction on song-control brain regions in fledgling (both sexes, 23-26 days old) song sparrows (Melospiza melodia). Food restriction selectively reduced HVC volume in both sexes. In addition, sex differences were evident in all three song-control regions. This study lends further support to a growing body of literature documenting a variety of behavioural, physiological and neural detriments in several songbird species resulting from early developmental stress.     

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     

Song and the song control pathway in the brain can develop independently of exposure to song in the sedge warbler

Previous studies have shown that female sedge warblers choose to mate with males that have more complex songs, and sexual selection has driven the evolution of both song complexity and the size of the major song control area (HVc) in the brain. In songbirds, learning from conspecifics plays a major role in song development and this study investigates the effects of isolation and exposure to song on song structure and the underlying song control system. Sibling pairs of hand-reared nestling sedge warblers were reared to sexual maturity under two conditions. Siblings in one group were reared individually in acoustic isolation in separate soundproof chambers. In the other group, siblings were reared together in an aviary with playback of recorded songs. The following spring, analysis of songs revealed that siblings reared in acoustic isolation produced normal song structures, including larger syllable repertoires than those exposed to song. We found no significant differences in the volumes of HVc, nucleus robustus archistnatalis, the lateral portion of the magnocellular nucleus and the density of dendritic spines between the two groups. Males exceeded females in all these measures, and also had a larger telencephalon. Our experiments show that complex song, sexual dimorphism in brain structure, and the size of song nuclei can all develop independently of exposure to song. These findings have important implications for how sexual selection can operate upon a complex male trait such as song and how it may also shape the more general evolution of brain structure in songbirds.     

Sexually size dimorphic brains and song complexity in passerine birds

Neural correlates of bird song involve the volume of particularsong nuclei in the brain that govern song development, production,and perception. Intra- and interspecific variation in the volumeof these song nuclei are associated with overall brain size,suggesting that the integration of complex songs into the brainrequires general neural augmentation. In a comparative studyof passerine birds based on generalized least square models,we tested this hypothesis by exploring the interspecific relationshipbetween overall brain size and repertoire size. We found nosignificant association between song complexity of males andbrain size adjusted for body size. However, species in whichmales produced complex songs tended to have sex differencesin overall brain size. This pattern became stronger when wecontrolled statistically for female song complexity by usingsex differences in song complexity. In species with large differencesin song complexity, females evolved smaller brains than didmales. Our results suggest no role for the evolution of extendedneural space, as reflected by total brain size, owing to songcomplexity. However, factors associated with sexual selectionmirrored by sex differences in song complexity were relatedto sexual dimorphism in overall brain size.     

Song and brain development in canaries raised under different conditions of acoustic and social isolation over two years

Early isolation experiments indicate that male songbirds learn their songs during an early sensitive period, although later work has shown that some open-ended learners modify songs in later years. Recent isolation experiments suggest that in some species song has a stronger genetic basis than previously thought. This study raised domestic canaries under different combinations of acoustic and social isolation and followed song development into the second year. Males raised alone in acoustic isolation developed songs with normal syllables, but larger repertoires and also produced syllables with lower repetition rates when compared to controls. The smallest repertoire occurred in males raised in a peer group. Isolate males had a smaller song control nucleus HVC than controls, but there was no effect on nucleus RA or on brain weight in general. In the second year, after introduction into a large normal colony, isolate and peer group males adjusted their syllable repertoire to normal size. In particular, the isolates reduced their repertoire even though the size of HVC showed a significant increase in volume. However, songs of isolate and peer group males still differ in repetition rate and number of single syllables in the common aviary. In contrast, control males showed low syllable turnover and no significant change in repertoire size. Nor did they show any significant change in the volumes of song control nuclei. It seems that complete isolation affects only some aspects of song and brain development, and later socialization corrects some but not all of these in the second year.     

Dynamic gene expression in the song system of zebra finches during the song learning period

The brain circuitry that controls song learning and production undergoes marked changes in morphology and connectivity during the song learning period in juvenile zebra finches, in parallel to the acquisition, practice and refinement of song. Yet, the genetic programs and timing of regulatory change that establish the neuronal connectivity and plasticity during this critical learning period remain largely undetermined. To address this question, we used in situ hybridization to compare the expression patterns of a set of 30 known robust molecular markers of HVC and/or area X, major telencephalic song nuclei, between adult and juvenile male zebra finches at different ages during development (20, 35, 50 days post‐hatch, dph). We found that several of the genes examined undergo substantial changes in expression within HVC or its surrounds, and/or in other song nuclei. They fit into broad patterns of regulation, including those whose expression within HVC during this period increases (COL12A1, COL 21A1, MPZL1, PVALB, and CXCR7) or decreases (e.g., KCNT2, SAP30L), as well as some that show decreased expression in the surrounding tissue with little change within song nuclei (e.g. SV2B, TAC1). These results reveal a broad range of molecular changes that occur in the song system in concert with the song learning period. Some of the genes and pathways identified are potential modulators of the developmental changes associated with the emergence of the adult properties of the song control system, and/or the acquisition of learned vocalizations in songbirds. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1315–1338, 2015     

Song repertoire size varies with HVC volume and is indicative of male quality in song sparrows (Melospiza melodia)

Complex birdsong is a classic example of a sexually selected ornamental trait. In many species, females prefer males with large song repertoires, possibly because repertoire size is limited by the size of song control nuclei which reflect developmental success. We investigated whether song repertoire size was indicative of brain area and male quality in song sparrows (Melospiza melodia) by determining if repertoire size was related to the volume of song control nucleus HVC, as well as several morphological, immunological and genetic indices of quality. We found that males with large repertoires had larger HVCs and were in better body condition. They also had lower heterophil to lymphocyte ratios, indicating less physiological stress and a robust immune system as measured by the number of lymphocytes per red blood cell. Song repertoire size also tended to increase with neutral-locus genetic diversity, as assessed by mean d2, but was not related to internal relatedness. Our results suggest several mechanisms that might explain the finding of a recent study that song sparrows with large song repertoires have higher lifetime fitness.     

白腰文鸟发声行为的神经发育

本文研究了 5～ 15 0日龄雄性白腰文鸟 (Lonchurastriataswinhoei)不同年龄段的声谱变化以及这种变化的神经调制机制。结果如下 :(1)HVC、RA和AreaX三个发声核团的神经联系基本接近成年鸟的水平后 ,幼鸟才开始学习鸣叫 (约 45日龄 ) ;(2 )HVC、RA和AreaX达到成年核团体积时 (约 80日龄 ) ,幼鸟才具有成年雄鸟的鸣叫模式 ;(3)发声控制核团的发育与核团间的神经支配有关 ,而基本不受鸣唱行为的影响 ,HVC、RA和AreaX的最快增长时间段各不相同 ,三个核团随年龄增长而呈现体积增长的显著变化 (one wayANOVA ,P <0 0 5 ) ,但各核团在任意两个时间段的体积差异并不都显著。结果提示 :发声行为产生的时间和发展与发声控制核团的发育、核团间的神经联系有关 ,最终的体积发育程度受内在遗传力的作用 ,同时可能还受神经核团建立正常神经联系时间的影响     

Distribution and onset of retinaldehyde dehydrogenase (zRalDH) expression in zebra finch brain: lack of sex difference in HVC and RA at early posthatch ages

Using in situ hybridization to detect the expression of the retinoic acid synthesizing enzyme (retinaldehyde dehydrogenase: zRalDH) mRNA, we mapped the distribution of its expression in adult zebra finch brain. In the neural song circuit, strong expression was found in high vocal center (HVC), para-HVC, and at a very low level in the robust nucleus of the arcopallium (RA). The expression in HVC and RA was found in both males and females. Outside of the song system, major areas of expression were in medial nidopallium (N), hyperpallium apicale (HA), mesopallium ventrale (MV), taenial amygdala (TnA), cerebellar Purkinje cells, and nucleus isthmo-opticus (IO). In nestlings, we found zRalDH mRNA expression in HVC and RA as early as posthatch day 4 or 5 (P4-5), although the expression varied among individuals. Thus, retinoic acid synthesis in HVC and RA could participate in song system formation and development. However, we found no sex difference in volume or intensity of zRalDH and androgen receptor (AR) expression in HVC and RA at P11 prior to the development of significant size dimorphisms in these nuclei. The size of HVC in females at P11 defined by zRalDH expression was greater than that in adult females, suggesting that HVC might experience net cell loss between P11 and adulthood.     

Syllable repertoire and the size of the song control system in captive canaries (Serinus canaria)

In songbirds, there is considerable interest in relationships between song structure and the size of the song control system in the forebrain. In male canaries, earlier studies have reported that repertoire size increased with age, and positive correlations were obtained between repertoire size and the volume of song control nuclei such as high vocal center (HVC). Here we investigate whether age has an effect upon both the song structure and the morphology of two song control nuclei [HVC and robustus archistriatalis (RA)] that are important in song production. We recorded songs from an aviary population of 1- and 2-year-old male domesticated canaries. We found that repertoire size, number of sexually attractive (sexy) syllables, and size of song nuclei did not differ between 1- and 2-year-old males. Neither did we find significant correlations between syllable repertoire size and the size of the song control nuclei. However, HVC size was positively correlated with the proportion of sexy syllables in the repertoires of 2-year-old males. Some older males may enhance vocal performance by modifying the control of syllables rather than by increasing repertoire size or neural space.     

Site-specific retinoic acid production in the brain of adult songbirds

The song system of songbirds, a set of brain nuclei necessary for song learning and production, has distinctive morphological and functional properties. Utilizing differential display, we searched for molecular components involved in song system regulation. We identified a cDNA (zRalDH) that encodes a class 1 aldehyde dehydrogenase. zRalDH was highly expressed in various song nuclei and synthesized retinoic acid efficiently. Brain areas expressing zRalDH generated retinoic acid. Within song nucleus HVC, only projection neurons not undergoing adult neurogenesis expressed zRalDH. Blocking zRalDH activity in the HVC of juveniles interfered with normal song development. Our results provide conclusive evidence for localized retinoic acid synthesis in an adult vertebrate brain and indicate that the retinoic acid-generating system plays a significant role in the maturation of a learned behavior.     

Effects of early song experience on song preferences and song control and auditory brain regions in female house finches (Carpodacus mexicanus)

We examined the effects of song tutoring on adult song preferences, volume of song-control brain regions, and activity of auditory brain regions in female house finches (Carpodacus mexicanus). Hand-reared females were tutored with local songs, foreign songs, or no song. We then examined adult song preferences, determined the Nissl-defined volume of the song-control nuclei HVc, Area X, and RA, and compared the number of cells immunoreactive for Zenk protein in the auditory regions NCM and cmHV, following playback of songs heard early in life (Tutor/Playback Match) versus not heard (Tutor/Playback Nonmatch). All hand-reared birds exhibited preferences for locally recorded song over foreign or heterospecific song. We found no difference in the volume of song-control nuclei among the three groups. As well, we found no difference in the number of Zenk immunoreactive cells in NCM and cmHV between females in the Tutor/Playback Match group and females in the Tutor/Playback Nonmatch group. Isolate-reared birds showed greater Zenk immunoreactivity following song playback than either tutored group. Thus, early auditory experience may not play a role in adult geographic song preferences, suggesting that genetic factors can lead to preferences for songs of local dialects. Song tutoring did not influence the size of song-control regions nor Zenk induction levels following song playback, suggesting that early experience with particular songs does not influence Zenk expression. However, overall greater activation in isolate females in auditory areas suggests that exposure to song early in life may increase the selectivity of Zenk activation to song playback in auditory areas.