Sexual equality in zebra finch song preference: evidence for a dissociation between song recognition and production learning

Song in oscine birds is a culturally inherited mating signal and sexually dimorphic. From differences in song production learning, sex differences in song recognition learning have been inferred but rarely put to a stringent test. In zebra finches, Taeniopygia guttata, females never sing and the species has one of the greatest neuroanatomical differences in song-related brain nuclei reported for songbirds. Preference tests with sibling groups for which exposure to song had been identical during the sensitive phase for song learning in males, revealed equally strong influence of the tutor's song (here the father) on males' and females' adult song preferences. Both sexes significantly preferred the father's over unfamiliar song when having free control over exposure to playbacks via an operant task. The sibling comparisons suggest that this preference developed independently of the song's absolute quality: variation between siblings was as great as between nests. The results show that early exposure has an equally strong influence on males' and females' song preferences despite the sexual asymmetry in song production learning. This suggests that the trajectory for song recognition learning is independent of the one for song production learning.     

Beak gape dynamics during song in the zebra finch

Bird song is a complex communication behavior that requires the coordination of several motor systems. Sound is produced in the syrinx and then modified by the upper vocal tract, but the specific nature and dynamics of this modification are not well understood. To determine the contribution of beak movements to sound modification, we studied the beak gape patterns in zebra finches (Taeniopygia guttata). Subsyringeal air sac pressure and song were recorded together with changes in beak gape, which were monitored with a magneto-sensitive transducer. Beak gape was positively correlated with fundamental frequency, peak frequency, and subsyringeal air sac pressure in all but one bird. For harmonic stacks, peak frequency increased with increasing beak gape, and the relationship between fundamental frequency and beak gape was no longer significant. Experimentally holding the beak open or closed had acoustic consequences consistent with the model in which beak movements change upper vocal tract length and, thus, the filter properties. Beak gape was positively correlated with sound amplitude in all but two birds. The relationship between beak aperture and amplitude may, however, be indirect because air sac pressure is correlated with amplitude and beak gape. The beak is opened quickly and to its widest aperture immediately prior to the onset of sound and at rapid transitions in sound, suggesting that beak movements may affect vibratory behavior of the labia.     

Tracheal length changes during zebra finch song and their possible role in upper vocal tract filtering

Sounds produced in the avian vocal organ may be modified by filter properties of the upper vocal tract. Possible mechanisms to actively control filter characteristics include movements of the beak, tongue, and larynx and adjustments of tracheal length. We investigated whether length changes of the trachea are a likely mechanism for adjusting upper vocal tract filter properties during song in the zebra finch (Taeniopygia guttata). Tracheal length was monitored at the basal end using sonomicrometry and was recorded together with subsyringeal air sac pressure and acoustic output. Tracheal shortening occurred at the onset of song bouts, and during each motif the tracheal length decreased during expiratory pressure pulses and increased during the short inspirations. A bilateral tracheal syringeal nerve cut confirmed that the initial shortening at the onset of the song bout is an active shortening of the trachea (i.e., mediated by syringeal muscle activity). The modulation of length during the motif was not affected by the denervation and is most likely driven by the pressurization of the interclavicular air sac. The absolute length change during the motif was small (<0.2 mm) and not clearly related to acoustic features of the song. For example, some high-frequency syllables, which are generated during inspiration, were accompanied by tracheal elongation. Because this elongation shifts tube resonances to lower frequencies, it is inconsistent with an active adjustment of length to enhance high frequency sounds. The small magnitude and inconsistent nature of dynamic tracheal length changes during song make it unlikely that they significantly affect vocal tract filter properties if the trachea is modeled as a rigid tube.     

Neuron loss and addition in developing zebra finch song nuclei are independent of auditory experience during song learning

In zebra finches early auditory experience is critical for normal song development. Young males first listen to and memorize a suitable song model and then use auditory feedback from their own vocalizations to mimic that model. During these two phases of vocal learning, song-related brain regions exhibit large, hormone-induced changes in volume and neuron number. Overlap between these neural changes and auditory-based vocal learning suggests that processing and acquiring auditory input may influence cellular processes that determine neuron number in the song system. We addressed this hypothesis by measuring neuron density, nuclear volume, and neuron number within the song system of normal male zebra finches and males deafened prior to song learning (10 days of age). Measures were obtained at 25, 50, 65, and 120 days of age, and included four song nuclei: the hyperstriatum ventralis pars caudalis or higher vocal center (HVc), Area X, the robust nucleus of the archistriatum (RA), and the lateral magnocellular nucleus of the anterior neostriatum (IMAN). In both HVc and Area X, nuclear volume and neuron number increased markedly with age in both normal and deafened birds. The volume of RA also increased with age and was not affected by early deafening. In IMAN, deafening also did not affect the overall age-related loss of neurons, although at 25 days neuron number was slightly less in deafened than in normal birds. We conclude that while the addition and loss of neurons in the developing song system may provide plasticity essential for song learning, these changes do not reflect learning.(ABSTRACT TRUNCATED AT 250 WORDS)     

Food for song: expression of c-Fos and ZENK in the zebra finch song nuclei during food aversion learning

Background

Specialized neural pathways, the song system, are required for acquiring, producing, and perceiving learned avian vocalizations. Birds that do not learn to produce their vocalizations lack telencephalic song system components. It is not known whether the song system forebrain regions are exclusively evolved for song or whether they also process information not related to song that might reflect their ‘evolutionary history’.

Methodology/Principal Findings

To address this question we monitored the induction of two immediate-early genes (IEGs) c-Fos and ZENK in various regions of the song system in zebra finches (Taeniopygia guttata) in response to an aversive food learning paradigm; this involves the association of a food item with a noxious stimulus that affects the oropharyngeal-esophageal cavity and tongue, causing subsequent avoidance of that food item. The motor response results in beak and head movements but not vocalizations. IEGs have been extensively used to map neuro-molecular correlates of song motor production and auditory processing. As previously reported, neurons in two pallial vocal motor regions, HVC and RA, expressed IEGs after singing. Surprisingly, c-Fos was induced equivalently also after food aversion learning in the absence of singing. The density of c-Fos positive neurons was significantly higher than that of birds in control conditions. This was not the case in two other pallial song nuclei important for vocal plasticity, LMAN and Area X, although singing did induce IEGs in these structures, as reported previously.

Conclusions/Significance

Our results are consistent with the possibility that some of the song nuclei may participate in non-vocal learning and the populations of neurons involved in the two tasks show partial overlap. These findings underscore the previously advanced notion that the specialized forebrain pre-motor nuclei controlling song evolved from circuits involved in behaviors related to feeding.     

Right-side dominance for song control in the zebra finch

Adult male zebra finches underwent unilateral denervation of the syrinx or unilateral lesion of the forebrain nucleus HVC known to be important for song control. Disruptive effects of song were greater after right-side than after left-side operations. After denervation of the right half of the syrinx, the fundamental frequencies of all syllables within a song converged on a value near 500 Hz, and nearly all syllables were altered in type. In contrast, the syllables produced after denervation of the left side of the syrinx largely maintained their preoperative frequencies, and fewer syllables changed in type. Unlike nerve sections, HVC lesions did not result in strikingly lateralized effects on syllable phonology; however, HVC lesions did affect the temporal patterning of a bird's song, whereas nerve sections did not, and changes in temporal patterning were more marked after right than after left HVC lesions. Right-side dominance for zebra finch song control is the reverse of that described in other songbird species with lateral asymmetry for vocal communication. We suggest that the need for a dominant side is more important than the side of dominance. © 1992 John Wiley & Sons, Inc.     

Towards quantification of vocal imitation in the zebra finch

The transition from an amorphous subsong into mature song requires a series of vocal changes. By tracing song elements during development, we have shown that the imitation trajectory to the target could not be predicted based on monotonic progression of vocal changes, indicating an internal component that imposes constraints on song development. Here we further examine the nature of constraints on song imitation in the zebra finch. We first present techniques for identifying and tracing distinctive vocal changes, and then we examine how sequences of vocal change are expressed and coordinated. Examples suggest two types of constraints on song imitation, based on the nature of the temporal context. Developmentally diachronic constraints are imposed by sequential dependencies between vocal changes as a function of developmental time, whereas developmentally synchronic constraints are given by the acoustic context of notes within the song. Finally, we show that the tendency of birds to copy certain sounds in the song model before others might be related to such constraints. We suggest that documenting the full range of distinctive vocal changes and the coordination of their expression would be useful for testing mechanisms of vocal imitation.     

Right-side dominance for song control in the zebra finch.

Adult male zebra finches underwent unilateral denervation of the syrinx or unilateral lesion of the forebrain nucleus HVC known to be important for song control. Disruptive effects on song were greater after right-side than after left-side operations. After denervation of the right half of the syrinx, the fundamental frequencies of all syllables within a song converged on a value near 500 Hz, and nearly all syllables were altered in type. In contrast, the syllables produced after denervation of the left side of the syrinx largely maintained their preoperative frequencies, and fewer syllables changed in type. Unlike nerve sections, HVC lesions did not result in strikingly lateralized effects on syllable phonology; however, HVC lesions did affect the temporal patterning of a bird's song, whereas nerve sections did not, and changes in temporal patterning were more marked after right than after left HVC lesions. Right-side dominance for zebra finch song control is the reverse of that described in other songbird species with lateral asymmetry for vocal communication. We suggest that the need for a dominant side is more important than the side of dominance.     

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.     

Respiratory units of motor production and song imitation in the zebra finch

Juvenile male zebra finches (Taeniopygia guttata) learn a stereotyped song by imitating sounds from adult male tutors. Their song is composed of a series of syllables, which are separated by silent periods. How acoustic units of song are translated into respiratory and syringeal motor gestures during the song learning process is not well understood. To learn about the respiratory contribution to the imitation process, we recorded air sac pressure in 38 male zebra finches and compared the acoustic structures and air sac pressure patterns of similar syllables qualitatively and quantitatively. Acoustic syllables correspond to expiratory pressure pulses and most often (74%) entire syllables are copied using similar air sac pressure patterns. Even notes placed within different syllables are generated with similar air sac pressure patterns when only segments of syllables are copied (9%). A few of the similar syllables (17%) are generated with a modified pressure pattern, typically involving addition or deletion of an inspiration. The high similarity of pressure patterns for like syllables indicates that generation of particular sounds is constrained to a narrow range of air sac pressure conditions. Following presentation of stroboscope flashes, song was typically interrupted at the end of an expiratory pressure pulse, confirming that expirations and, therefore, syllables are the smallest unit of motor production of song. Silent periods, which separate syllables acoustically, are generated by switching from expiration to inspiration. Switching between respiratory phases, therefore, appears to play a dominant role in organizing the stereotyped motor program for song production.     

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     

Dietary retinoic acid affects song maturation and gene expression in the song system of the zebra finch

Vitamin A, an essential nutrient, is required in its acidic form (retinoic acid) for normal embryogenesis and neuronal development, typically within well-defined concentration ranges. In zebra finches, a songbird species, localized retinoic acid synthesis in the brain is important for the development of song, a learned behavior sharing significant commonalities with speech acquisition in humans. We tested how dietary retinoic acid affects the development of song behavior and the brain's system for song control. Supplemental doses of retinoic acid given to juveniles during the critical period for song learning resulted in more variable or plastic-like songs when the birds reached adulthood, compared to the normal songs of vehicle-fed controls. We also observed that several genes (brinp1, nrgn, rxr-alpha, and sdr2/scdr9) had altered levels of expression in specific nuclei of the song system when comparing the experimental and control diet groups. Interestingly, we found significant correlations between gene expression levels in nuclei of the anterior forebrain pathway (lMAN and area X) and the degree of variability in the recorded songs. We observed, however, no major morphological effects such as changes in the volumes of song nuclei. Overall, our results lend further support to a fundamental role of retinoic acid in song maturation and point to possible molecular pathways associated with this action. The data also demonstrate that dietary content of Vitamin A can affect the maturation of a naturally learned complex behavior.     

Paradoxical hypermasculinization of the zebra finch song system by an antiestrogen

Exogenous estrogens, when administered to hatchling female zebra finches, masculinize the morphology and function of their neural vocal control system. The first of two experiments evaluated whether tamoxifen citrate is an antiestrogen in zebra finches, and the second determined whether it would block the masculinization hypothesized to be caused in hatchling males by the males' endogenous estradiol. In the first experiment adult female zebra finches were ovariectomized and injected for 10 days with estradiol benzoate (EB), tamoxifen, EB and tamoxifen combined, or vehicle (control). The dependent variable was oviduct weight. The EB-stimulated growth of the oviduct was blocked by tamoxifen, which had no effects when administered alone. Thus, tamoxifen acts as an antiestrogen in the zebra finch oviduct. In Experiment 2, male and female zebra finches were treated with tamoxifen or vehicle for the first 20 days after hatching. The males were castrated at 20 days. At 60 days we compared the song control regions of experimental and control males and females. In both sexes tamoxifen increased the somatic areas of neurons in RA (robust nucleus of the archistriatum), HVc (caudal nucleus of the ventral hyperstriatum), and MAN (magnocellular nucleus of the anterior neostriatum). Tamoxifen also increased the volumes of HVc, RA, MAN, and Area X in males. Thus, tamoxifen failed to block masculinization of males, but masculinized females and hypermasculinized males. Tamoxifen's hypermasculinization of the male and masculinization of the female song system is paradoxical given that (1) estradiol does not have similar effects on the male song system, and (2) tamoxifen antagonizes the effects of EB in the oviduct.     

Tamoxifen's effects on the zebra finch song system are estrogenic,not antiestrogenic

Antiestrogens fail to block the masculine ontogeny of the zebra finch song system that is hypothesized to occur as a result of early estrogen action. Moreover, they hypermasculinize the male, and masculinize the female song systems. In experiment 1, we assessed whether these antiestrogenic effects might mimic estrogenic actions. Zebra finch chicks received one of two treatments. They were given estradiol benzoate (EB) or vehicle daily for the first 20 days after hatching and sacrificed at 60 days of age, or they received EB or vehicle for the first 25 days after hatching, at which time they were sacrificed. In the day 60 group, certain attributes of the song system were hypermasculinized in males and masculinized in females by EB, when compared with controls. In the day 25 group, males treated with EB were partially demasculinized, while the females were partially masculinized. In experiment 2, we assessed whether simultaneous treatment with tamoxifen was capable of antagonizing the effects of EB obtained in experiment 1 (day 60 group). Sixty-day-old females, previously treated with both EB and tamoxifen for the first 20 days after hatching, had more masculine song regions than females treated with either EB alone or tamoxifen alone. In males, the effects of the combined treatment of EB and tamoxifen over those produced by tamoxifen alone were not as dramatic as in the female. These results are similar to those obtained in systems where tamoxifen is purely estrogenic and suggest that in the song system, tamoxifen acts as an estrogen, not an antiestrogen.     

Developmental stress selectively affects the song control nucleus HVC in the zebra finch

Songbirds sing complex songs as a result of evolution through sexual selection. The evolution of such sexually selected traits requires genetic control, as well as selection on their expression. Song is controlled by a discrete neural pathway in the brain, and song complexity has been shown to correlate with the volume of specific song control nuclei. As such, the development of these nuclei, in particular the high vocal centre (HVC), is thought to be the mechanism controlling signal expression indicating male quality. We tested the hypothesis that early developmental stress selectively affects adult HVC size, compared with other brain nuclei. We did this by raising cross-fostered zebra finches (Taeniopygia guttata) under stressed and controlled conditions and determining the effect on adult HVC size. Our results confirm the strong influence of environmental conditions, particularly on HVC development, and therefore on the expression of complex songs. The results also show that both environmental and genetic factors affect the development of several brain nuclei, highlighting the developmental plasticity of the songbird brain. In all, these results explain how the complex song repertoires of songbirds can evolve as honest indicators of male quality.     

Basal forebrain cholinergic modulation of auditory activity in the zebra finch song system

The cholinergic basis of auditory "gating" in the sensorimotor nucleus HVc and its efferent target robustus archistriatalis (RA) was investigated in anesthetized zebra finches. Injections of cholinergic agonists carbachol or muscarine into HVc strongly affected discharge rates and diminished auditory responsiveness in both HVc and its target RA, changes toward an awake-like condition. HVc nicotine injections produced similar strong effects in HVc, but weaker and inconsistent effects in RA. Stimulation of basal forebrain (BF) produced an initial transient network shutdown followed by diminished auditory responsiveness in HVc and RA. All stimulation effects were blocked when preceded by HVc injections of nicotinic or muscarinic antagonists. Thus, BF cholinergic modulation of song system auditory activity acting via functionally distinct HVc circuits can contribute to auditory gating. We hypothesize that wakeful BF activity levels block sensory input to motor systems and adaptively change during behavior to allow sensorimotor feedback such as auditory feedback during singing.     

Tamoxifen's effects on the zebra finch song system are estrogenic, not antiestrogenic.

Antiestrogens fail to block the masculine ontogeny of the zebra finch song system that is hypothesized to occur as a result of early estrogen action. Moreover, they hypermasculinize the male, and masculinize the female song systems. In experiment 1, we assessed whether these antiestrogenic effects might mimic estrogenic actions. Zebra finch chicks received one of two treatments. They were given estradiol benzoate (EB) or vehicle daily for the first 20 days after hatching and sacrificed at 60 days of age, or they received EB or vehicle for the first 25 days after hatching, at which time they were sacrificed. In the day 60 group, certain attributes of the song system were hypermasculinized in males and masculinized in females by EB, when compared with controls. In the day 25 group, males treated with EB were partially demasculinized, while the females were partially masculinized. In experiment 2, we assessed whether simultaneous treatment with tamoxifen was capable of antagonizing the effects of EB obtained in experiment 1 (day 60 group). Sixty-day-old females, previously treated with both EB and tamoxifen for the first 20 days after hatching, had more masculine song regions than females treated with either EB alone or tamoxifen alone. In males, the effects of the combined treatment of EB and tamoxifen over those produced by tamoxifen alone were not as dramatic as in the female. These results are similar to those obtained in systems where tamoxifen is purely estrogenic and suggest that in the song system, tamoxifen acts as an estrogen, not an antiestrogen.