Song Feature Specific Analysis of Isolate Song Reveals Interspecific Variation in Learned Components

Studies of avian vocal development without exposure to conspecific song have been conducted in many passerine species, and the resultant isolate song is often interpreted to represent an expression of the genetic code for conspecific song. There is wide recognition that vocal learning exists in oscine songbirds, but vocal learning has only been thoroughly investigated in a few model species, resulting in a narrow view of birdsong learning. By extracting acoustic signals from published spectrograms, we have reexamined the findings of isolate studies with a universally applicable semi‐automated quantitative analysis regimen. When song features were analyzed in light of three different production aspects (respiratory, syringeal, and central programming of sequence), all three show marked interspecific variability in how close isolate song features are to normal. This implies that song learning mechanisms are more variable than is commonly recognized. Our results suggest that the interspecific variation shows no readily observable pattern reflecting phylogeny, which has implications for understanding the mechanisms behind the evolution of avian vocal communication. We emphasize that song learning in passerines provides an excellent opportunity to investigate the evolution of a complex, plastic trait from a phylogenetic perspective.     

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.     

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 effect of syringeal deafferentation on the stimulated vocalization in the domestic chick (Gallus gallus)

The r?le of syringeal feedback on the electrically elicited vocalization in chickens (Gallus gallus) was studied. The vocalization patterns elicited by stimulating the low-threshold "calling areas" in the midbrain, was examined before and after syringeal deafferentation obtained by cutting the X-XII anastomosis on either the left and right side. The results show that section of the X-XII anastomosis on the left side produces consistently a clear-cut lowering of the threshold for vocalization and an increase of the amplitude of the individual calls with no change in the repetition rate of the call sequences. All these effects are enhanced when the contralateral anastomosis is also severed. These results show that the bilateral syringeal deafferentation does not change the overall song pattern performance, that remains stereotyped both in duration and complexity, consistent with the hypothesis that, in adult birds, stable song patterns are not dependent on peripheral sources of feedback, but are ruled by a learned central control program. However the modification of vocalization threshold and amplitude suggests the hypothesis that the syringeal feedback plays an inhibitory r?le on vocalization by modulating the excitability of the central structures involved in the vocalization activity.     

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.     

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.     

Phonation in the Orange-winged Amazon parrot,Amazona amazonica

Summary The syrinx of the Orange-winged Amazon parrot includes two external tympaniform membranes thought to be involved in sound production. The position of these membranes at the confluence of the bronchial and tracheal lumina requires that during phonation they be driven by a single column of air and by its attending turbulence patterns. Because of this anatomical arrangement, the phonatory output of either right or left syringeal half is grossly affected by denervation of the ipsilateral or contralateral syringial muscles. Following unilateral syringeal denervation the unbalanced oscillation of the two external tympaniform membranes generates noise. Form this we may infer that normally the parrot syrinx acts as a unitary sound source. Syringeal innervation is provided by the tracheosyringealis branch of the hypoglossus nerve. Each tracheosyringealis innervates both syringeal halves. Section of either the right or left tracheosyringealis leads to a minor and temporary change in the structure of vocalization. One week after the operation the vocalizations are delivered as pre-operatively. There is no indication of either right or left hypoglossal dominance in the phonatory control of the parrot syrinx. Other observations presented here are used to speculate on the possible role of the parrot tongue in altering the resonating properties of the nasopharyngeal space and generating speech like formants.     

Contributions of syringeal muscles to respiration and vocalization in the zebra finch

Acute and chronic electromyographic (EMG) recordings from individual syringeal muscles were used to study syringeal participation in respiration and vocalization. In anesthetized birds, all syringeal muscles recorded were active to some degree during the expiratory phase of respiration, following activity in the abdominal musculature and preceding the emergence of breath from the nostril. In awake birds, the ventralis (V) muscle fired a strong, consistent burst, but the dorsalis (D) was variable both in strength and timing. Denervation of V is sufficient to produce the wheezing respiration originally seen in birds with complete bilateral section of the tracheosyringeal nerve. Complete syringeal denervation also removed almost all the acoustic features that distinguish individual song syllables, but had a minor effect on the temporal structure of song. When activity in V and D was recorded in awake, vocalizing birds, D was active before and during sound production, and V showed a small burst before sound onset and a vigorous burst timed to the termination of sound. During song, V was consistently active at sound offset, but also participated during sound for narrow bandwidth syllables. For some syllables (simple harmonic stacks), neither muscle was active. These data suggest that V contributes to syllable termination during vocalization and may silence the syrinx during normal respiration. D contributes to the acoustic structure of most syllables, and V may contribute to a special subset of syllables. In summary, the syringeal muscles show different activity patterns during respiration and vocalization and can be independently activated during vocalization, depending on the syllable produced.     

Sexual dimorphism and bilateral asymmetry of syrinx and vocal tract in the European starling (Sturnus vulgaris)

Sexually dimorphic vocal behavior in zebra finches (Taeniopygia guttata) is associated with a 100% larger syrinx in males and other morphological adaptations of the sound source. The songbird syrinx consists of two independent sound sources, whose specialization for different spectral ranges may be reflected in morphological properties, but the morphology of labia and syringeal skeleton have not been investigated for lateralized specializations. Similarly, little is known whether the morphology of the songbird vocal tract reflects differences in vocal behavior. Here, we tested the hypothesis that different vocal behavior and specialization is reflected in the morphology. We investigated syringeal and upper vocal tract morphology of male and female European starlings (Sturnus vulgaris). Female starlings exhibit smaller vocal repertoires and sing at lower rates than males. In males, the left syrinx produces mostly low frequencies, while the right one is used for higher notes. Macroscopic and histological techniques were used to record nineteen measurements from the syrinx and the vocal tract which were tested for sexual differences in syrinx and vocal tract and for lateral asymmetry within the syrinx. Sexually dimorphic vocal behavior is reflected in the morphology of the starling syrinx. Males have a larger syrinx with the size difference attributable to increased muscle mass and three enlarged elements of the syringeal skeleton. The upper vocal tract, however, does not differ between males and females. Distinct lateralization was found in two elements of the syringeal skeleton of females, and the labia in the left syrinx are larger than those on the right in both sexes. The sexual dimorphism of the syringeal size is smaller in starlings (35%) than in zebra finches (100%), which is consistent with the different vocal behavior of females in both species. The morphological differences between the two sound sources are discussed in relation to their vocal performance. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Lateralization and motor stereotype of song production in the brown-headed cowbird

Song production in adult brown-headed cowbirds(Molothrus ater ater) is lateralized, with a slight right syringeal dominance. The left size of the syrinx produces low-frequency (200–2000 Hz) notes within the introductory note clusters, while the right side produces the higher-frequency (1500–6000 Hz) introductory notes, the interphrase unit (10–12 kHz), and the final high-frequency whistle (5–13 kHz). Cross-correlation analyses reveal that individual cowbirds produce each of their four to seven song types with a distinct stereotyped motor pattern–as judged by the patterns of syringeal airflow and subsyringeal pressure. The acoustic differences across song types are reflected in the differences in the bronchial airflow and air sac pressure patterns associated with song production. These motor differences are particularly striking within the second and third introductory note clusters where there is a rapid switching back and forth between the two sides of the syrinx in the production of notes. These motor skills may be especially important in producing behaviorally effective song. 1994 John Wiley & Sons, Inc.     

Electromyographic studies of the syrinx in parrots (Aves,Psittacidae)

Summary The vocal organ (syrinx) of a bird may contain either extrinsic muscles alone or both extrinsic and intrinsic muscles. The former arise and insert on the trachea and affect the syrinx only indirectly; the latter also arise on the trachea but insert directly on syringeal elements. It is widely supposed that syringeal muscles can affect modulations of the sounds the birds make, and further, that the intrinsic muscles are closely associated with such a function. However, the exact roles of the two groups of muscles have not been directly observed.The psittacid syrinx, which has one (for practical purposes) pair of extrinsic and two pairs of intrinsic muscles, is about as simple as one can find in birds capable of uttering a wide variety of sounds. We have taken electromyograms from the syringeal muscles of five species of parrots. In all of these, the extrinsic sternotrachealis showed the simple activation pattern activity previously described from several non-passerine species that possess only extrinsic muscles. The intrinsic muscles, however, showed a variety of activity patterns. The relatively simple call of Cyanoliseus patagonus again showed the simple activation pattern. In Myiopsitta monachus, the muscles showed a string of pulses that matched to pulses of sound in a strongly amplitude modulated call. Agapornis roseicollis used at least two distinct patterns, each associated with a different call.The results are consistent with an hypothesis that, because of their indirect attachment of the syrinx, extrinsic muscles are poorly suited to the production of precise, rapid changes in syringeal action, but rather will function in an on-off switch capacity. Intrinsic muscles are so situated that, given proper neurological stimulus, they can effect a variety of alterations in the sound pattern. Hence, intrinsic muscles are necessary for the evolution of large vocabularies and variable vocal behavior.     

鸣禽发声器官在鸣啭过程中的功能

鸣禽的鸣啭是一种习得行为,与人类的学习过程较为相似.因此鸣禽作为一种动物模型在研究人类学习记忆方面得到广泛的应用.鸣管和鸣肌是鸣禽鸣啭的主要器官,对鸣啭过程起着复杂的调节作用.此外,不同的鸣禽在鸣啭时,其发声器官具有不同的侧别优势.对近年在鸣禽发声器官功能方面的研究进行综述.     

Bilaterally symmetrical respiratory activity during lateralized birdsong

We investigated whether activity of expiratory muscles reflects lateralized activity of the vocal organ during production of birdsong. Respiration and syringeal motor activity were assessed in brown thrashers by monitoring bilateral airflow and subsyringeal air sac pressure, together with the electromyographic activity of expiratory abdominal muscles and vocal output. Activity of expiratory muscles was always present on both sides, regardless of whether song was produced bilaterally or on only one side of the syrinx. The average amplitude of expiratory EMG of one side does not change significantly, even if that side is silent during phonation. The temporal pattern of the electromyogram (EMG) was similar on both sides. Bilateral bursts of EMG activity on both sides accompanied changes in the rate of syringeal airflow, even when these flow fluctuations were generated only by one side of the syrinx. Motor commands to the respiratory muscles therefore appear to be bilaterally distributed, in contrast to the lateralized motor control of the syrinx.     

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.     

Lateralization of syringeal function during song production in the canary

The canary (Serinus canaria) vocal organ, the syrinx, has two separate sound sources, one in the cranial end of each bronchus. Previous investigations of whether song syllables are produced unilaterally or bilaterally have provided two contradictory results, as one researcher suggested that almost all syllables are produced by the left side of the syrinx alone, whereas another researcher suggested that both sides contribute similarly to all syllables. Our experiments, which involved unilateral bronchus plugging followed later by denervation of the ipsilateral syringeal muscles, attempted to resolve this disagreement. The males with right bronchus plugs, singing on the left side of the syrinx alone, produced nearly normal songs, whereas the birds with left bronchus plugs, singing on the right side, sang quite poorly. Interpretation of these data is difficult because it is not clear how syringeal function would be affected if the airflow rate through the intact side is increased above normal, nor is it known if the bird can compensate for bronchus occlusion. Nonetheless, we suggest that in male canaries most syllables are normally sung by the left side alone, with some syllables being produced by the right side alone and some being sung by both sides together. Right nerve section had little effect on the right-bronchus-plugged males' ability to sing, but the repertoires of the left-plugged males were altered after left nerve section, indicating the possibility that signals carried by the left nerve exert an influence on the contralateral side.     

Superfast vocal muscles control song production in songbirds

Birdsong is a widely used model for vocal learning and human speech, which exhibits high temporal and acoustic diversity. Rapid acoustic modulations are thought to arise from the vocal organ, the syrinx, by passive interactions between the two independent sound generators or intrinsic nonlinear dynamics of sound generating structures. Additionally, direct neuromuscular control could produce such rapid and precisely timed acoustic features if syringeal muscles exhibit rare superfast muscle contractile kinetics. However, no direct evidence exists that avian vocal muscles can produce modulations at such high rates. Here, we show that 1) syringeal muscles are active in phase with sound modulations during song over 200 Hz, 2) direct stimulation of the muscles in situ produces sound modulations at the frequency observed during singing, and that 3) syringeal muscles produce mechanical work at the required frequencies and up to 250 Hz in vitro. The twitch kinematics of these so-called superfast muscles are the fastest measured in any vertebrate muscle. Superfast vocal muscles enable birds to directly control the generation of many observed rapid acoustic changes and to actuate the millisecond precision of neural activity into precise temporal vocal control. Furthermore, birds now join the list of vertebrate classes in which superfast muscle kinetics evolved independently for acoustic communication.     

Motor dynamics of song production by mimic thrushes

In brown thrashers (Toxostoma rufum) and grey catbirds (Dumetella carolinensis) neither side of the syrinx has a consistently dominant role in song production. During song, the two sides operate independently, but in close cooperation with each other and with the respiratory muscles which are capable of adjusting expiratory effort to maintain a constant rate of syringeal airflow despite sudden changes in syringeal resistance. Phonation is frequently switched from one side of the syrinx to the other, both between syllables and within a syllable. When both sides of the syrinx produce sound simultaneously, their respective contributions are seldom harmonically related. The resulting “two-voice” syllables sometimes contain difference tones with prominent sinusoidal amplitude modulation (AM). Rarely, both sides simultaneously produce the same sound. In general, however, the frequency range of sound contributed by the right syrinx is higher than that of the left syrinx. The right syrinx is also primarily responsible for producing a rapid cyclical amplitude modulation which is a characteristic feature of some syllables. This kind of AM is generated by either repetitive brief bursts of sound from the right side that modulate the amplitude of a continuous sound arising on the left side or cyclically opening the right syrinx, allowing unmodulated expiratory air to bypass the phonating left side. 1994 John Wiley & Sons, Inc.     

Behavioural and morphological dose-responses to testosterone and to 5α-dihydrotestosterone in the castrated male Japanese quail

Morphological and behavioural effects of testosterone (T) and of 5 α-dihydrotestosterone (5α-DHT) injected daily for a 3-week period at dosages of 0.5, 1, 5 and 10 mg for T and 1 and 5 mg for α-DHT were studied in the adult male castrated Japanese quail. Injections of 0.5 or 1.0 mg T produced only slight development of the cloacal gland while the other four treatments stimulated growth which reached maximal or submaximal values. Testosterone injections stimulated sexual activities; some such effects were also observed after treatment with 5α-DHT. Although both steroids elicited crowing, there were qualitative differences between quails given 5α-DHT and those given T and intacts. These differences were not due to the development of the sternotracheal (syringeal) muscles, the weights of which were increased and reached similar values in the 5α-DHT and T (5 and 10 mg) treated males. These results are discussed in the context of our present knowledge of the mechanisms of regulation of reproduction processes by testosterone and its metabolites in birds.     

Mechanisms of thermoregulation in plants

Endothermic heating of floral tissues and even thermoregulation is known to occur in a number of plant species across a wide taxonomic range. The mechanisms by which flowers heat, however, are only just beginning to be understood, and even less is known about how heating is regulated in response to changes in ambient temperature. We have recently demonstrated that the alternative pathway of respiration, in which the alternative oxidase (AOX) rather than cytochrome C (COX) acts as terminal electron acceptor, is responsible for heat generation in one thermoregulating species, the sacred lotus (Nelumbo nucifera). In the March issue of the Journal of Experimental Botany we further demonstrated that AOX-mediated heat production in this species is regulated at both the level of gene expression and also post-translationally. Similarly, AOX has also been implicated in heat production in other thermogenic species. In this addendum we discuss the central role of AOX in heat production and how post-translational mechanisms may provide the fine control necessary for thermoregulation.Key words: alternative oxidase, Nelumbo nucifera, thermogenic plants, uncoupling proteins