Songbirds use pulse tone register in two voices to generate low-frequency sound

The principal physical mechanism of sound generation is similar in songbirds and humans, despite large differences in their vocal organs. Whereas vocal fold dynamics in the human larynx are well characterized, the vibratory behaviour of the sound-generating labia in the songbird vocal organ, the syrinx, is unknown. We present the first high-speed video records of the intact syrinx during induced phonation. The syrinx of anaesthetized crows shows a vibration pattern of the labia similar to that of the human vocal fry register. Acoustic pulses result from short opening of the labia, and pulse generation alternates between the left and right sound sources. Spontaneously calling crows can also generate similar pulse characteristics with only one sound generator. Airflow recordings in zebra finches and starlings show that pulse tone sounds can be generated unilaterally, synchronously or by alternating between the two sides. Vocal fry-like dynamics therefore represent a common production mechanism for low-frequency sounds in songbirds. These results also illustrate that complex vibration patterns can emerge from the mechanical properties of the coupled sound generators in the syrinx. The use of vocal fry-like dynamics in the songbird syrinx extends the similarity to this unusual vocal register with mammalian sound production mechanisms.     

Sexual Dimorphism of the Zebra Finch Syrinx Indicates Adaptation for High Fundamental Frequencies in Males

Background

In many songbirds the larger vocal repertoire of males is associated with sexual dimorphism of the vocal control centers and muscles of the vocal organ, the syrinx. However, it is largely unknown how these differences are translated into different acoustic behavior.

Methodology/Principal Findings

Here we show that the sound generating structures of the syrinx, the labia and the associated cartilaginous framework, also display sexual dimorphism. One of the bronchial half rings that position and tense the labia is larger in males, and the size and shape of the labia differ between males and females. The functional consequences of these differences were explored by denervating syringeal muscles. After denervation, both sexes produced equally low fundamental frequencies, but the driving pressure generally increased and was higher in males. Denervation strongly affected the relationship between driving pressure and fundamental frequency.

Conclusions/Significance

The syringeal modifications in the male syrinx, in concert with dimorphisms in neural control and muscle mass, are most likely the foundation for the potential to generate an enhanced frequency range. Sexually dimorphic vocal behavior therefore arises from finely tuned modifications at every level of the motor cascade. This sexual dimorphism in frequency control illustrates a significant evolutionary step towards increased vocal complexity in birds.     

Muscle‐dependent and hormone‐dependent differentiation of the vocal control premotor nucleus robustus archistriatalis and the motornucleus hypoglossus pars tracheosyringealis of the zebra finch

Sex differences in the vertebrate brain (brain sex) are thought to develop owing to the tissue specific action of gonadal hormones similar to the development of secundary sex characteristics of the body. Small sex differences in body anatomy could, however, retrogradely control the sexual differentiation of the central nervous system. This possibility has so far been verified only for motorneuron pools, since the connectivity of sex‐specific higher brain areas to the sexual dimorphic periphery is frequently not well known. Here, we tested whether somatic sex differences feed back on higher brain areas by bilateral denervation of the syringeal musculature of zebra finches before, during, and after onset of estrogen‐sensitive sexual differentiation of forebrain vocal nuclei such as RA (nucleus robustus archistriatalis). In the zebra finch, the sound‐producing musculature (the syrinx), the syrinx motornucleus hypolossus pars tracheosyringealis (nXIIts), and the RA are much larger in males compared to females. Tract tracing studies revealed that the volume and neuron size distribution of the nXIIts was sexually dimorphic in intact but not in animals denervated as juveniles. In contrast, the volume of RA and size of RA neurons of denervated animals were highly sexually dimorphic. Furthermore, estrogen masculinized the RA of denervated females. Thus, sexual differentiation of the RA but not of the nXIIts appears independent of somatic sex differences. The syrinx muscles are, however, important for the soma size of those RA neurons that project to the nXIIts. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 220–231, 2000     

The acoustic effect of vocal tract adjustments in zebra finches

Vocal production in songbirds requires the control of the respiratory system, the syrinx as sound source and the vocal tract as acoustic filter. Vocal tract movements consist of beak, tongue and hyoid movements, which change the volume of the oropharyngeal–esophageal cavity (OEC), glottal movements and tracheal length changes. The respective contributions of each movement to filter properties are not completely understood, but the effects of this filtering are thought to be very important for acoustic communication in birds. One of the most striking movements of the upper vocal tract during vocal behavior in songbirds involves the OEC. This study measured the acoustic effect of OEC adjustments in zebra finches by comparing resonance acoustics between an utterance with OEC expansion (calls) and a similar utterance without OEC expansion (respiratory sounds induced by a bilateral syringeal denervation). X-ray cineradiography confirmed the presence of an OEC motor pattern during song and call production, and a custom-built Hall-effect collar system confirmed that OEC expansion movements were not present during respiratory sounds. The spectral emphasis during zebra finch call production ranging between 2.5 and 5 kHz was not present during respiratory sounds, indicating strongly that it can be attributed to the OEC expansion.     

Early Administration of 17β-Estradiol Partially Masculinizes Song Control Regions and α2-Adrenergic Receptor Distribution in European Starlings (Sturnus vulgaris)

The vocal control system in many songbird species is a sexually dimorphic neural circuit that mediates learning and production of song. The mechanism by which this system is sexually differentiated has been investigated in only one species, the zebra finch (Taeniopygia guttata). Estradiol may be involved in the sexual differentiation of this system, as female zebra finches treated with estradiol as nestlings develop a male-like song system; however, blocking estradiol action in embryonic and nestling male zebra finches does not demasculinize the song system. Therefore, the role of estradiol in song system development is unclear. The role of estradiol in song system sexual differentiation was assessed in European starlings (Sturnus vulgaris). This species is of potential interest because it is less extreme in the degree of sexual dimorphism of the song system and song behavior than zebra finches. While in the field, starling nestlings were implanted with 500 μg of estradiol at 3 days of age. These birds were brought into the laboratory at Day 11 and hand-reared. In females, estradiol produces significant increases in the volumes of song control regions defined by Nissl stain, as well as by autoradiography for α₂-adrenergic receptors; however, these estradiol-treated females have song systems that more closely resemble those of control females than control males. Estradiol-treated males exhibit significant hypermasculinization at 210 days of age, but this effect is transient and hypermasculinization is no longer evident at Day 345. The role of estradiol in sexual differentiation of the neural circuit mediating song behavior remains enigmatic.     

Muscle-dependent and hormone-dependent differentiation of the vocal control premotor nucleus robustus archistriatalis and the motornucleus hypoglossus pars tracheosyringealis of the zebra finch

Sex differences in the vertebrate brain (brain sex) are thought to develop owing to the tissue specific action of gonadal hormones similar to the development of secondary sex characteristics of the body. Small sex differences in body anatomy could, however, retrogradely control the sexual differentiation of the central nervous system. This possibility has so far been verified only for motorneuron pools, since the connectivity of sex-specific higher brain areas to the sexual dimorphic periphery is frequently not well known. Here, we tested whether somatic sex differences feed back on higher brain areas by bilateral denervation of the syringeal musculature of zebra finches before, during, and after onset of estrogen-sensitive sexual differentiation of forebrain vocal nuclei such as RA (nucleus robustus archistriatalis). In the zebra finch, the sound-producing musculature (the syrinx), the syrinx motornucleus hypoglossus pars tracheosyringealis (nXIIts), and the RA are much larger in males compared to females. Tract tracing studies revealed that the volume and neuron size distribution of the nXIIts was sexually dimorphic in intact but not in animals denervated as juveniles. In contrast, the volume of RA and size of RA neurons of denervated animals were highly sexually dimorphic. Furthermore, estrogen masculinized the RA of denervated females. Thus, sexual differentiation of the RA but not of the nXIIts appears independent of somatic sex differences. The syrinx muscles are, however, important for the soma size of those RA neurons that project to the nXIIts.     

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.     

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.     

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.     

New perspectives on mechanisms of sound generation in songbirds

The physical mechanisms of sound generation in the vocal organ, the syrinx, of songbirds have been investigated mostly with indirect methods. Recent direct endoscopic observation identified vibrations of the labia as the principal sound source. This model suggests sound generation in a pulse-tone mechanism similar to human phonation with the labia forming a pneumatic valve. The classical avian model proposed that vibrations of the thin medial tympaniform membranes are the primary sound generating mechanism. As a direct test of these two hypotheses we ablated the medial tympaniform membranes in two species (cardinal and zebra finch) and found that both were still able to phonate and sing without functional membranes. Small changes in song structure (harmonic emphasis, frequency control) occurred after medial tympaniform membrane ablation and suggest that the medial tympaniform membranes play a role in adjusting tension on the labia. Such a role is consistent with the fact that the medial tympaniform membranes are directly attached to the medial labia. There is no experimental support for a third hypothesis, proposing an aerodynamic model for generation of tonal sounds. Indirect tests (song in heliox atmosphere) as well as direct (labial vibration during tonal sound) measurements of syringeal vibrations support a vibration-based sound-generating mechanism even for tonal sounds.     

Peripheral control and lateralization of birdsong

Recent studies on several species of oscine songbirds show that they achieve their varied vocal performances through coordinated activity of respiratory, syringeal, and other vocal tract muscles in ways that take maximum advantage of the acoustic flexibility made possible by the presence of two independently controlled sound sources in their bipartite syrinx (vocal organ). During song, special motor programs to respiratory muscles alter the pattern of ventilation to maintain the supply of respiratory air and oxygen to permit songs of long duration, high syllable repetition rates, or maximum spectral complexity. Each side of the syrinx receives its own motor program that, together with that sent to respiratory muscles, determines the acoustic properties of the ipsilaterally produced sound. The acoustic expression of these bilaterally distinct, phonetic motor patterns depends on the action of dorsal syringeal adductor muscles that, by opening or closing the ipsilateral side of the syrinx to airflow, determine the amount each side contributes to song. The syringeally generated sound is further modified by muscles that control the shape of the vocal tract. Different species have adopted different motor strategies that use the left and right sides of the syrinx in patterns of unilateral, bilateral, alternating, or sequential phonation to achieve the differing temporal and spectral characteristics of their songs. As a result, the degree of song lateralization probably varies between species to form a continuum from unilateral dominance to bilateral equality. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 632–652, 1997     

Perineuronal nets and vocal plasticity in songbirds: A proposed mechanism to explain the difference between closed‐ended and open‐ended learning

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience‐dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open‐ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin‐positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed‐ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin‐positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed‐ended and open‐ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975–994, 2017     

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.     

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.     

Singing Under the Influence: Examining the Effects of Nutrition and Addiction on a Learned Vocal Behavior

The songbird model is widely established in a number of laboratories for the investigation of the neurobiology and development of vocal learning. While vocal learning is rare in the animal kingdom, it is a trait that songbirds share with humans. The neuroanatomical and physiological organization of the brain circuitry that controls learned vocalizations has been extensively characterized, particularly in zebra finches (Taeniopygia guttata). Recently, several powerful molecular and genomic tools have become available in this organism, making it an attractive choice for neurobiologists interested in the neural and genetic basis of a complex learned behavior. Here, we briefly review some of the main features of vocal learning and associated brain structures in zebra finches and comment on some examples that illustrate how themes related to nutrition and addiction can be explored using this model organism.     

The anatomy of the syrinx in passerine birds

The macroscopical structure of the organ of voice in songbirds has long been known, but detailed information on the microscopical anatomy of the syrinx has generally been lacking. Observations based largely on macroscopical evidence have led to a number of erroneous interpretations of function of various syringeal components, and lacking microscopical information, the vocal mechanism of birds cannot be adequately understood.
A wide variety of passeriform bird syrinxes have been studied by means of serial sections. Although there is much less variation in syringeal anatomy amongst songbirds than there is in the other orders of birds, and although all songbird syrinxes conform to the same basic pattern, there is nevertheless marked variation in various syringeal components between different passerine groups. Variations in syringeal structure within families Corvidae ( Corvus corone, C. frugilegus ), Sturnidae ( Sturnus vulgaris, Gracula religiosa ), Turdidae ( Turdus merula, Erithacus rubecula ), Hirundinidae ( Delichon urbica ), Ploceidae ( Passer domesticus ) and Paridae ( Parus major, Aegithalos caudatus ) are described and discussed. The significance of these findings in relation to bird sound production is discussed.     

The anatomy of vocal divergence in North American Elk and European red deer

Loud and frequent vocalizations play an important role in courtship behavior in Cervus species. European red deer (Cervus elaphus) produce low‐pitched calls, whereas North American elk (Cervus canadensis) produce high‐pitched calls, which is remarkable for one of the biggest land mammals. Both species engage their vocal organs in elaborate maneuvers but the precise mechanism is unknown. Vocal organs were compared by macroscopic and microscopic dissection. The larynx is sexually dimorphic in red deer but not in elk. The laryngeal lumen is more constricted in elk, and narrows further during ontogeny. Several elements of the hyoid skeleton and two of four vocal tract segments are longer in red deer than in elk allowing greater vocal tract expansion and elongation. We conclude that elk submit the larynx and vocal tract to much higher tension than red deer, whereby, enormously stressed vocal folds of reduced effective length create a high resistance glottal source. The narrow, high impedance laryngeal vestibulum matches glottal and vocal tract impedance allowing maximum power transfer. In red deer longer and relaxed vocal folds create a less resistant glottal source and a wider vestibulum matches the low glottal impedance to the vocal tract, thereby also ensuring maximum power transfer. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

The neuromuscular control of birdsong.

Birdsong requires complex learned motor skills involving the coordination of respiratory, vocal organ and craniomandibular muscle groups. Recent studies have added to our understanding of how these vocal subsystems function and interact during song production. The respiratory rhythm determines the temporal pattern of song. Sound is produced during expiration and each syllable is typically followed by a small inspiration, except at the highest syllable repetition rates when a pattern of pulsatile expiration is used. Both expiration and inspiration are active processes. The oscine vocal organ, the syrinx, contains two separate sound sources at the cranial end of each bronchus, each with independent motor control. Dorsal syringeal muscles regulate the timing of phonation by adducting the sound-generating labia into the air stream. Ventral syringeal muscles have an important role in determining the fundamental frequency of the sound. Different species use the two sides of their vocal organ in different ways to achieve the particular acoustic properties of their song. Reversible paralysis of the vocal organ during song learning in young birds reveals that motor practice is particularly important in late plastic song around the time of song crystallization in order for normal adult song to develop. Even in adult crystallized song, expiratory muscles use sensory feedback to make compensatory adjustments to perturbations of respiratory pressure. The stereotyped beak movements that accompany song appear to have a role in suppressing harmonics, particularly at low frequencies.     

Behavioral discrimination of sexually dimorphic calls by male zebra finches requires an intact vocal motor pathway

Vocal communication between zebra finches includes the exchange of long calls (LCs) as well as song. By using this natural call behavior and quantifying the LCs emitted in response to playbacks of LCs of other birds, we have previously shown that adult male zebra finches have a categorical preference for the LCs of females over those of males. Female LCs are acoustically simpler than male LCs, which include complex acoustic features that are learned during development. Production of these male-typical features requires an intact nucleus RA, the sexually dimorphic source of the main telencephalic projection to brainstem vocal effectors. We have now made bilateral lesions of RA in 17 adult males and tested their discrimination behavior in the call response situation. Lesioned birds continue to call, but lose the male-typical preference for female LCs. The degree of loss is correlated with the extent of RA damage. Further, the simplified LCs of males with RA lesions have a variable duration that is correlated with stimulus features. In effect, the call response behavior of lesioned males becomes like that of females. Apparently, in the absence of RA, the remaining intact structures receive different call information than RA normally does, and/or process it differently. This suggests that the vocal motor nucleus RA could play a role in the transformation of a signal encoding the salience of stimulus parameters into a control signal that modulates the probability and strength of responding.     

Mechanisms of song production in the Australian magpie

Australian magpies (Gymnorhina tibicen) are notable for their vocal prowess. We investigated the syringeal and respiratory dynamics of vocalization by two 6-month-old males, whose songs had a number of adult features. There was no strong lateral syringeal dominance and unilateral phonation was most often achieved by closing the syringeal valve on the contralateral side of the syrinx. Unlike other songbirds studied, magpies sometimes used an alternative syringeal motor pattern during unilateral phonation in which both sides of the syrinx are partially adducted and open to airflow. Also, in contrast to most other songbirds, the higher fundamental frequency during two-voice syllables was usually generated on the left side of the syrinx. Amplitude modulation, a prominent feature of magpie song, was produced by linear or nonlinear interactions between different frequencies which may originate either on opposite sides of the syrinx or on the same side. Pulse tones, similar to vocal fry in human speech, were present in some calls. Unlike small songbirds, the fundamental of the modal frequency can be as low as that of the pulse tone, suggesting that large birds may have evolved pulse tones to increase acoustic diversity, rather than decrease the fundamental frequency.