BackgroundLike 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.ResultsTo 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.ConclusionsOur 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.

As animals vocalize, their vocal organ transforms motor commands into vocalizations for social communication. In birds, the physical mechanisms by which vocalizations are produced and controlled remain unresolved because of the extreme difficulty in obtaining in vivo measurements. Here, we introduce an ex vivo preparation of the avian vocal organ that allows simultaneous high-speed imaging, muscle stimulation and kinematic and acoustic analyses to reveal the mechanisms of vocal production in birds across a wide range of taxa. Remarkably, we show that all species tested employ the myoelastic-aerodynamic (MEAD) mechanism, the same mechanism used to produce human speech. Furthermore, we show substantial redundancy in the control of key vocal parameters ex vivo, suggesting that in vivo vocalizations may also not be specified by unique motor commands. We propose that such motor redundancy can aid vocal learning and is common to MEAD sound production across birds and mammals, including humans.

Heterozygous disruptions of the Forkhead transcription factor FoxP2 impair acquisition of speech and language. Experimental downregulation in brain region Area X of the avian ortholog FoxP2 disrupts song learning in juvenile male zebra finches. In vitro, transcriptional activity of FoxP2 requires dimerization with itself or with paralogs FoxP1 and FoxP4. Whether this is the case in vivo is unknown. To provide the means for future functional studies we cloned FoxP4 from zebra finches and compared regional and cellular coexpression of FoxP1, FoxP2, and FoxP4 mRNA and protein in brains of juvenile and adult male zebra finches. In the telencephalic song nuclei HVC, RA, and Area X, the three investigated FoxPs were either expressed alone or occurred in specific combinations with each other, as shown by double in situ hybridization and triple immunohistochemistry. FoxP1 and FoxP4 but not FoxP2 were expressed in RA and in the HVCRA and HVCX projection neurons. In Area X and the surrounding striatum the density of neurons expressing all three FoxPs together or FoxP1 and FoxP4 together was significantly higher than the density of neurons expressing other combinations. Interestingly, the proportions of Area X neurons expressing particular combinations of FoxPs remained constant at all ages. In addition, FoxP-expressing neurons in adult Area X express dopamine receptors 1A, 1B, and 2. Together, these data provide the first evidence that Area X neurons can coexpress all avian FoxP subfamily members, thus allowing for a variety of regulatory possibilities via heterodimerization that could impact song behavior in zebra finches.

The biomechanics of sound production forms an integral part of the neuromechanical control loop of avian vocal motor control. However, we critically lack quantification of basic biomechanical parameters describing the vocal organ, the syrinx, such as material properties of syringeal elements, forces and torques exerted on, and motion of the syringeal skeleton during song. Here, we present a novel marker-based 3D stereoscopic imaging technique to reconstruct 3D motion of servo-controlled actuation of syringeal muscle insertions sites in vitro and focus on two muscles controlling sound pitch. We furthermore combine kinematic analysis with force measurements to quantify elastic properties of sound producing medial labia (ML). The elastic modulus of the zebra finch ML is 18 kPa at 5% strain, which is comparable to elastic moduli of mammalian vocal folds. Additionally ML lengthening due to musculus syringealis ventralis (VS) shortening is intrinsically constraint at maximally 12% strain. Using these values we predict sound pitch to range from 350–800 Hz by VS modulation, corresponding well to previous observations. The presented methodology allows for quantification of syringeal skeleton motion and forces, acoustic effects of muscle recruitment, and calibration of computational birdsong models, enabling experimental access to the entire neuromechanical control loop of vocal motor control.