Fast Retrograde Access to Projection Neuron Circuits Underlying Vocal Learning in Songbirds.

Understanding the structure and function of neural circuits underlying speech and language is a vital step toward better treatments for diseases of these systems. Songbirds, among the few animal orders that share with humans the ability to learn vocalizations from a conspecific, have provided many insights into the neural mechanisms of vocal development. However, research into vocal learning circuits has been hindered by a lack of tools for rapid genetic targeting of specific neuron populations to meet the quick pace of developmental learning. Here, we present a viral tool that enables fast and efficient retrograde access to projection neuron populations. In zebra finches, Bengalese finches, canaries, and mice, we demonstrate fast retrograde labeling of cortical or dopaminergic neurons. We further demonstrate the suitability of our construct for detailed morphological analysis, for in vivo imaging of calcium activity, and for multi-color brainbow labeling.

In situ vocal fold properties and pitch prediction by dynamic actuation of the songbird syrinx.

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.

Differential coexpression of FoxP1, FoxP2, and FoxP4 in the Zebra Finch (Taeniopygia guttata) song system.

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 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.