Defects in Ultrasonic Vocalization of Cadherin-6 Knockout Mice

Background

Although some molecules have been identified as responsible for human language disorders, there is still little information about what molecular mechanisms establish the faculty of human language. Since mice, like songbirds, produce complex ultrasonic vocalizations for intraspecific communication in several social contexts, they can be good mammalian models for studying the molecular basis of human language. Having found that cadherins are involved in the vocal development of the Bengalese finch, a songbird, we expected cadherins to also be involved in mouse vocalizations.

Methodology/Principal Findings

To examine whether similar molecular mechanisms underlie the vocalizations of songbirds and mammals, we categorized behavioral deficits including vocalization in cadherin-6 knockout mice. Comparing the ultrasonic vocalizations of cadherin-6 knockout mice with those of wild-type controls, we found that the peak frequency and variations of syllables were differed between the mutant and wild–type mice in both pup-isolation and adult-courtship contexts. Vocalizations during male-male aggression behavior, in contrast, did not differ between mutant and wild–type mice. Open-field tests revealed differences in locomotors activity in both heterozygote and homozygote animals and no difference in anxiety behavior.

Conclusions/Significance

Our results suggest that cadherin-6 plays essential roles in locomotor activity and ultrasonic vocalization. These findings also support the idea that different species share some of the molecular mechanisms underlying vocal behavior.     

Comparative Analysis of Protocadherin-11 X-Linked Expression among Postnatal Rodents,Non-Human Primates,and Songbirds Suggests Its Possible Involvement in Brain Evolution

Background

Protocadherin-11 is a cell adhesion molecule of the cadherin superfamily. Since, only in humans, its paralog is found on the Y chromosome, it is expected that protocadherin-11X/Y plays some role in human brain evolution or sex differences. Recently, a genetic mutation of protocadherin-11X/Y was reported to be associated with a language development disorder. Here, we compared the expression of protocadherin-11 X-linked in developing postnatal brains of mouse (rodent) and common marmoset (non-human primate) to explore its possible involvement in mammalian brain evolution. We also investigated its expression in the Bengalese finch (songbird) to explore a possible function in animal vocalization and human language faculties.

Methodology/Principal Findings

Protocadherin-11 X-linked was strongly expressed in the cerebral cortex, hippocampus, amygdala and brainstem. Comparative analysis between mice and marmosets revealed that in certain areas of marmoset brain, the expression was clearly enriched. In Bengalese finches, protocadherin-11 X-linked was expressed not only in nuclei of regions of the vocal production pathway and the tracheosyringeal hypoglossal nucleus, but also in areas homologous to the mammalian amygdala and hippocampus. In both marmosets and Bengalese finches, its expression in pallial vocal control areas was developmentally regulated, and no clear expression was seen in the dorsal striatum, indicating a similarity between songbirds and non-human primates.

Conclusions/Significance

Our results suggest that the enriched expression of protocadherin-11 X-linked is involved in primate brain evolution and that some similarity exists between songbirds and primates regarding the neural basis for vocalization.     

Syringeal specialization of frequency control during song production in the Bengalese finch (Lonchura striata domestica)

Background

Singing in songbirds is a complex, learned behavior which shares many parallels with human speech. The avian vocal organ (syrinx) has two potential sound sources, and each sound generator is under unilateral, ipsilateral neural control. Different songbird species vary in their use of bilateral or unilateral phonation (lateralized sound production) and rapid switching between left and right sound generation (interhemispheric switching of motor control). Bengalese finches (Lonchura striata domestica) have received considerable attention, because they rapidly modify their song in response to manipulations of auditory feedback. However, how the left and right sides of the syrinx contribute to acoustic control of song has not been studied.

Methodology

Three manipulations of lateralized syringeal control of sound production were conducted. First, unilateral syringeal muscular control was eliminated by resection of the left or right tracheosyringeal portion of the hypoglossal nerve, which provides neuromuscular innervation of the syrinx. Spectral and temporal features of song were compared before and after lateralized nerve injury. In a second experiment, either the left or right sound source was devoiced to confirm the role of each sound generator in the control of acoustic phonology. Third, air pressure was recorded before and after unilateral denervation to enable quantification of acoustic change within individual syllables following lateralized nerve resection.

Significance

These experiments demonstrate that the left sound source produces louder, higher frequency, lower entropy sounds, and the right sound generator produces lower amplitude, lower frequency, higher entropy sounds. The bilateral division of labor is complex and the frequency specialization is the opposite pattern observed in most songbirds. Further, there is evidence for rapid interhemispheric switching during song production. Lateralized control of song production in Bengalese finches may enhance acoustic complexity of song and facilitate the rapid modification of sound production following manipulations of auditory feedback.     

Temporal recalibration in vocalization induced by adaptation of delayed auditory feedback

Background

We ordinarily perceive our voice sound as occurring simultaneously with vocal production, but the sense of simultaneity in vocalization can be easily interrupted by delayed auditory feedback (DAF). DAF causes normal people to have difficulty speaking fluently but helps people with stuttering to improve speech fluency. However, the underlying temporal mechanism for integrating the motor production of voice and the auditory perception of vocal sound remains unclear. In this study, we investigated the temporal tuning mechanism integrating vocal sensory and voice sounds under DAF with an adaptation technique.

Methods and Findings

Participants produced a single voice sound repeatedly with specific delay times of DAF (0, 66, 133 ms) during three minutes to induce ‘Lag Adaptation’. They then judged the simultaneity between motor sensation and vocal sound given feedback. We found that lag adaptation induced a shift in simultaneity responses toward the adapted auditory delays. This indicates that the temporal tuning mechanism in vocalization can be temporally recalibrated after prolonged exposure to delayed vocal sounds. Furthermore, we found that the temporal recalibration in vocalization can be affected by averaging delay times in the adaptation phase.

Conclusions

These findings suggest vocalization is finely tuned by the temporal recalibration mechanism, which acutely monitors the integration of temporal delays between motor sensation and vocal sound.     

Comparative genomics reveals molecular features unique to the songbird lineage

Background

Songbirds (oscine Passeriformes) are among the most diverse and successful vertebrate groups, comprising almost half of all known bird species. Identifying the genomic innovations that might be associated with this success, as well as with characteristic songbird traits such as vocal learning and the brain circuits that underlie this behavior, has proven difficult, in part due to the small number of avian genomes available until recently. Here we performed a comparative analysis of 48 avian genomes to identify genomic features that are unique to songbirds, as well as an initial assessment of function by investigating their tissue distribution and predicted protein domain structure.

Results

Using BLAT alignments and gene synteny analysis, we curated a large set of Ensembl gene models that were annotated as novel or duplicated in the most commonly studied songbird, the Zebra finch (Taeniopygia guttata), and then extended this analysis to 47 additional avian and 4 non-avian genomes. We identified 10 novel genes uniquely present in songbird genomes. A refined map of chromosomal synteny disruptions in the Zebra finch genome revealed that the majority of these novel genes localized to regions of genomic instability associated with apparent chromosomal breakpoints. Analyses of in situ hybridization and RNA-seq data revealed that a subset of songbird-unique genes is expressed in the brain and/or other tissues, and that 2 of these (YTHDC2L1 and TMRA) are highly differentially expressed in vocal learning-associated nuclei relative to the rest of the brain.

Conclusions

Our study reveals novel genes unique to songbirds, including some that may subserve their unique vocal control system, substantially improves the quality of Zebra finch genome annotations, and contributes to a better understanding of how genomic features may have evolved in conjunction with the emergence of the songbird lineage.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1082) contains supplementary material, which is available to authorized users.     

Dynamics of Vocalization-Induced Modulation of Auditory Cortical Activity at Mid-utterance

Background

Recent research has addressed the suppression of cortical sensory responses to altered auditory feedback that occurs at utterance onset regarding speech. However, there is reason to assume that the mechanisms underlying sensorimotor processing at mid-utterance are different than those involved in sensorimotor control at utterance onset. The present study attempted to examine the dynamics of event-related potentials (ERPs) to different acoustic versions of auditory feedback at mid-utterance.

Methodology/Principal findings

Subjects produced a vowel sound while hearing their pitch-shifted voice (100 cents), a sum of their vocalization and pure tones, or a sum of their vocalization and white noise at mid-utterance via headphones. Subjects also passively listened to playback of what they heard during active vocalization. Cortical ERPs were recorded in response to different acoustic versions of feedback changes during both active vocalization and passive listening. The results showed that, relative to passive listening, active vocalization yielded enhanced P2 responses to the 100 cents pitch shifts, whereas suppression effects of P2 responses were observed when voice auditory feedback was distorted by pure tones or white noise.

Conclusion/Significance

The present findings, for the first time, demonstrate a dynamic modulation of cortical activity as a function of the quality of acoustic feedback at mid-utterance, suggesting that auditory cortical responses can be enhanced or suppressed to distinguish self-produced speech from externally-produced sounds.     

Comparative analysis of mineralocorticoid receptor expression among vocal learners (Bengalese finch and budgerigar) and non-vocal learners (quail and ring dove) has implications for the evolution of avian vocal learning

Mineralocorticoid receptor is the receptor for corticosteroids such as corticosterone or aldosterone. Previously, we found that mineralocorticoid receptor was highly expressed in song nuclei of a songbird, Bengalese finch (Lonchura striata var. domestica). Here, to examine the relationship between mineralocorticoid receptor expression and avian vocal learning, we analyzed mineralocorticoid receptor expression in the developing brain of another vocal learner, budgerigar (Melopsittacus undulatus) and non-vocal learners, quail (Coturnix japonica) and ring dove (Streptopelia capicola). Mineralocorticoid receptor showed vocal control area-related expressions in budgerigars as Bengalese finches, whereas no such mineralocorticoid receptor expressions were seen in the telencephalon of non-vocal learners. Thus, these results suggest the possibility that mineralocorticoid receptor plays a role in vocal development of parrots as songbirds and that the acquisition of mineralocorticoid receptor expression is involved in the evolution of avian vocal learning.     

The zebra finch neuropeptidome: prediction,detection and expression

Background  

Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions.     

Functional MRI of auditory responses in the zebra finch forebrain reveals a hierarchical organisation based on signal strength but not selectivity

Background

Male songbirds learn their songs from an adult tutor when they are young. A network of brain nuclei known as the ‘song system’ is the likely neural substrate for sensorimotor learning and production of song, but the neural networks involved in processing the auditory feedback signals necessary for song learning and maintenance remain unknown. Determining which regions show preferential responsiveness to the bird''s own song (BOS) is of great importance because neurons sensitive to self-generated vocalisations could mediate this auditory feedback process. Neurons in the song nuclei and in a secondary auditory area, the caudal medial mesopallium (CMM), show selective responses to the BOS. The aim of the present study is to investigate the emergence of BOS selectivity within the network of primary auditory sub-regions in the avian pallium.

Methods and Findings

Using blood oxygen level-dependent (BOLD) fMRI, we investigated neural responsiveness to natural and manipulated self-generated vocalisations and compared the selectivity for BOS and conspecific song in different sub-regions of the thalamo-recipient area Field L. Zebra finch males were exposed to conspecific song, BOS and to synthetic variations on BOS that differed in spectro-temporal and/or modulation phase structure. We found significant differences in the strength of BOLD responses between regions L2a, L2b and CMM, but no inter-stimuli differences within regions. In particular, we have shown that the overall signal strength to song and synthetic variations thereof was different within two sub-regions of Field L2: zone L2a was significantly more activated compared to the adjacent sub-region L2b.

Conclusions

Based on our results we suggest that unlike nuclei in the song system, sub-regions in the primary auditory pallium do not show selectivity for the BOS, but appear to show different levels of activity with exposure to any sound according to their place in the auditory processing stream.     

Song Practice Promotes Acute Vocal Variability at a Key Stage of Sensorimotor Learning

Background

Trial by trial variability during motor learning is a feature encoded by the basal ganglia of both humans and songbirds, and is important for reinforcement of optimal motor patterns, including those that produce speech and birdsong. Given the many parallels between these behaviors, songbirds provide a useful model to investigate neural mechanisms underlying vocal learning. In juvenile and adult male zebra finches, endogenous levels of FoxP2, a molecule critical for language, decrease two hours after morning song onset within area X, part of the basal ganglia-forebrain pathway dedicated to song. In juveniles, experimental ‘knockdown’ of area X FoxP2 results in abnormally variable song in adulthood. These findings motivated our hypothesis that low FoxP2 levels increase vocal variability, enabling vocal motor exploration in normal birds.

Methodology/Principal Findings

After two hours in either singing or non-singing conditions (previously shown to produce differential area X FoxP2 levels), phonological and sequential features of the subsequent songs were compared across conditions in the same bird. In line with our prediction, analysis of songs sung by 75 day (75d) birds revealed that syllable structure was more variable and sequence stereotypy was reduced following two hours of continuous practice compared to these features following two hours of non-singing. Similar trends in song were observed in these birds at 65d, despite higher overall within-condition variability at this age.

Conclusions/Significance

Together with previous work, these findings point to the importance of behaviorally-driven acute periods during song learning that allow for both refinement and reinforcement of motor patterns. Future work is aimed at testing the observation that not only does vocal practice influence expression of molecular networks, but that these networks then influence subsequent variability in these skills.     

The songbird syrinx morphome: a three-dimensional, high-resolution, interactive morphological map of the zebra finch vocal organ

Background

Like human infants, songbirds learn their species-specific vocalizations through imitation learning. The birdsong system has emerged as a widely used experimental animal model for understanding the underlying neural mechanisms responsible for vocal production learning. However, how neural impulses are translated into the precise motor behavior of the complex vocal organ (syrinx) to create song is poorly understood. First and foremost, we lack a detailed understanding of syringeal morphology.

Results

To fill this gap we combined non-invasive (high-field magnetic resonance imaging and micro-computed tomography) and invasive techniques (histology and micro-dissection) to construct the annotated high-resolution three-dimensional dataset, or morphome, of the zebra finch (Taeniopygia guttata) syrinx. We identified and annotated syringeal cartilage, bone and musculature in situ in unprecedented detail. We provide interactive three-dimensional models that greatly improve the communication of complex morphological data and our understanding of syringeal function in general.

Conclusions

Our results show that the syringeal skeleton is optimized for low weight driven by physiological constraints on song production. The present refinement of muscle organization and identity elucidates how apposed muscles actuate different syringeal elements. Our dataset allows for more precise predictions about muscle co-activation and synergies and has important implications for muscle activity and stimulation experiments. We also demonstrate how the syrinx can be stabilized during song to reduce mechanical noise and, as such, enhance repetitive execution of stereotypic motor patterns. In addition, we identify a cartilaginous structure suited to play a crucial role in the uncoupling of sound frequency and amplitude control, which permits a novel explanation of the evolutionary success of songbirds.     

Behaviours Associated with Acoustic Communication in Nile Tilapia (Oreochromis niloticus)

Background

Sound production is widespread among fishes and accompanies many social interactions. The literature reports twenty-nine cichlid species known to produce sounds during aggressive and courtship displays, but the precise range in behavioural contexts is unclear. This study aims to describe the various Oreochromis niloticus behaviours that are associated with sound production in order to delimit the role of sound during different activities, including agonistic behaviours, pit activities, and reproduction and parental care by males and females of the species.

Methodology/Principal Findings

Sounds mostly occur during the day. The sounds recorded during this study accompany previously known behaviours, and no particular behaviour is systematically associated with sound production. Males and females make sounds during territorial defence but not during courtship and mating. Sounds support visual behaviours but are not used alone. During agonistic interactions, a calling Oreochromis niloticus does not bite after producing sounds, and more sounds are produced in defence of territory than for dominating individuals. Females produce sounds to defend eggs but not larvae.

Conclusion/Significance

Sounds are produced to reinforce visual behaviours. Moreover, comparisons with O. mossambicus indicate two sister species can differ in their use of sound, their acoustic characteristics, and the function of sound production. These findings support the role of sounds in differentiating species and promoting speciation. They also make clear that the association of sounds with specific life-cycle roles cannot be generalized to the entire taxa.     

Vocal Tract Articulation in Zebra Finches

Background

Birdsong and human vocal communication are both complex behaviours which show striking similarities mainly thought to be present in the area of development and learning. Recent studies, however, suggest that there are also parallels in vocal production mechanisms. While it has been long thought that vocal tract filtering, as it occurs in human speech, only plays a minor role in birdsong there is an increasing number of studies indicating the presence of sound filtering mechanisms in bird vocalizations as well.

Methodology/Principal Findings

Correlating high-speed X-ray cinematographic imaging of singing zebra finches (Taeniopygia guttata) to song structures we identified beak gape and the expansion of the oropharyngeal-esophageal cavity (OEC) as potential articulators. We subsequently manipulated both structures in an experiment in which we played sound through the vocal tract of dead birds. Comparing acoustic input with acoustic output showed that OEC expansion causes an energy shift towards lower frequencies and an amplitude increase whereas a wide beak gape emphasizes frequencies around 5 kilohertz and above.

Conclusion

These findings confirm that birds can modulate their song by using vocal tract filtering and demonstrate how OEC and beak gape contribute to this modulation.     

Development of Auditory-Vocal Perceptual Skills in Songbirds

Songbirds are one of the few groups of animals that learn the sounds used for vocal communication during development. Like humans, songbirds memorize vocal sounds based on auditory experience with vocalizations of adult “tutors”, and then use auditory feedback of self-produced vocalizations to gradually match their motor output to the memory of tutor sounds. In humans, investigations of early vocal learning have focused mainly on perceptual skills of infants, whereas studies of songbirds have focused on measures of vocal production. In order to fully exploit songbirds as a model for human speech, understand the neural basis of learned vocal behavior, and investigate links between vocal perception and production, studies of songbirds must examine both behavioral measures of perception and neural measures of discrimination during development. Here we used behavioral and electrophysiological assays of the ability of songbirds to distinguish vocal calls of varying frequencies at different stages of vocal learning. The results show that neural tuning in auditory cortex mirrors behavioral improvements in the ability to make perceptual distinctions of vocal calls as birds are engaged in vocal learning. Thus, separate measures of neural discrimination and behavioral perception yielded highly similar trends during the course of vocal development. The timing of this improvement in the ability to distinguish vocal sounds correlates with our previous work showing substantial refinement of axonal connectivity in cortico-basal ganglia pathways necessary for vocal learning.     

Auditory feedback control of vocal pitch during sustained vocalization: a cross-sectional study of adult aging

Background

Auditory feedback has been demonstrated to play an important role in the control of voice fundamental frequency (F₀), but the mechanisms underlying the processing of auditory feedback remain poorly understood. It has been well documented that young adults can use auditory feedback to stabilize their voice F₀ by making compensatory responses to perturbations they hear in their vocal pitch feedback. However, little is known about the effects of aging on the processing of audio-vocal feedback during vocalization.

Methodology/Principal Findings

In the present study, we recruited adults who were between 19 and 75 years of age and divided them into five age groups. Using a pitch-shift paradigm, the pitch of their vocal feedback was unexpectedly shifted ±50 or ±100 cents during sustained vocalization of the vowel sound/u/. Compensatory vocal F₀ response magnitudes and latencies to pitch feedback perturbations were examined. A significant effect of age was found such that response magnitudes increased with increasing age until maximal values were reached for adults 51–60 years of age and then decreased for adults 61–75 years of age. Adults 51–60 years of age were also more sensitive to the direction and magnitude of the pitch feedback perturbations compared to younger adults.

Conclusion

These findings demonstrate that the pitch-shift reflex systematically changes across the adult lifespan. Understanding aging-related changes to the role of auditory feedback is critically important for our theoretical understanding of speech production and the clinical applications of that knowledge.     

The role of auditory feedback in vocal learning and maintenance

Auditory experience is critical for the acquisition and maintenance of learned vocalizations in both humans and songbirds. Despite the central role of auditory feedback in vocal learning and maintenance, where and how auditory feedback affects neural circuits important to vocal control remain poorly understood. Recent studies of singing birds have uncovered neural mechanisms by which feedback perturbations affect vocal plasticity and also have identified feedback-sensitive neurons at or near sites of auditory and vocal motor interaction. Additionally, recent studies in marmosets have underscored that even in the absence of vocal learning, vocalization remains flexible in the face of changing acoustical environments, pointing to rapid interactions between auditory and vocal motor systems. Finally, recent studies show that a juvenile songbird's initial auditory experience of a song model has long-lasting effects on sensorimotor neurons important to vocalization, shedding light on how auditory memories and feedback interact to guide vocal learning.     

Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members

Cadherins play an important role in specific cell-cell adhesion events. Their expression appears to be tightly regulated during development and each tissue or cell type shows a characteristic pattern of cadherin molecules. Inappropriate regulation of their expression levels or functionality has been observed in human malignancies, in many cases leading to aggravated cancer cell invasion and metastasis. The cadherins form a superfamily with at least six subfamilies, which can be distinguished on the basis of protein domain composition, genomic structure, and phylogenetic analysis of the protein sequences. These subfamilies comprise classical or type-I cadherins, atypical or type-II cadherins, desmocollins, desmogleins, protocadherins and Flamingo cadherins. In addition, several cadherins clearly occupy isolated positions in the cadherin superfamily (cadherin-13, -15, -16, -17, Dachsous, RET, FAT, MEGF1 and most invertebrate cadherins). We suggest a different evolutionary origin of the protocadherin and Flamingo cadherin genes versus the genes encoding desmogleins, desmocollins, classical cadherins, and atypical cadherins. The present phylogenetic analysis may accelerate the functional investigation of the whole cadherin superfamily by allowing focused research of prototype cadherins within each subfamily.