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. 2017 Sep 12;7(1):11296.
doi: 10.1038/s41598-017-11258-1.

In Situ Vocal Fold Properties and Pitch Prediction by Dynamic Actuation of the Songbird Syrinx

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Free PMC article

In Situ Vocal Fold Properties and Pitch Prediction by Dynamic Actuation of the Songbird Syrinx

Daniel N Düring et al. Sci Rep. .
Free PMC article

Abstract

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.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Syrinx morphology. (A) Schematic sagittal cross-section through the syrinx showing the syringeal skeleton (grey) with the paired soft tissue masses LL and MVM (yellow). Muscles are omitted for clarity. (BD) Micro-computed tomography scan of the zebra finch syrinx (modified from ref. 30) showing bones (grey), cartilaginous pads (orange), muscles (pink) and sound producing MVM (yellow). (B) Ventral and dorsal view with the two muscles (red outlines) that directly attach on cartilaginous pads embedded in the medial labium, VS and MDS. (C) Medial view on the MVM with only soft tissues of right bronchus visible and left bronchus removed for clarity. The tympanum is the bony cylinder that is formed of fused tracheal rings. The pessulus is an ossified medial dorso-ventral bridge of the tympanum dividing the two bronchi. (D) Top view of panel C. For a detailed description of the zebra finch syrinx including 3D interactive PDF figures see ref. . Abbreviations: B1-3, bronchial bones 1-3; LDC, latero-dorsal cartilage; LL, lateral labium; MDC, medio-dorsal cartilage; MDS, musculus syringealis dorsalis medialis; ML, medial labium; MVC, medio-ventral cartilage; MVM, medial vibratory mass; PES, pessulus; TYMP, tympanum; VS, musculus syringealis ventralis.
Figure 2
Figure 2
Experimental set-up. (A) Sideview of the right MVM (yellow) in relation to the syringeal skeleton (grey) with the left bronchus removed for clarity. Carbon markers (black dots labelled 1–3) were placed on the MVM to quantify its deformation. The lines of action of the force vectors the VS and MDS muscles exert are indicated (Fvs and FMDS). (B) Schematic drawing of the experimental set-up. The syrinx is fixed in the experimental chamber to a tracheal connector, consisting of rigid polyethylene tubing, and bronchial connector, consisting of silicon tubing. The lever arm of the ergometer is placed outside the chamber and connected to the cartilage (here MVC) of the syrinx through a single 10-0 suture that is threaded through a small hole in the chamber (suture port). A 5 mm right angle prism is positioned with a micromanipulator to view the markers from two different angles. The red lined box indicates view in panel C. The inset (right) indicates where B1-3 were glued to the tympanum (blue) to fix them in the same coordinate system. (C) Top (left) and mirrored side view (right) of the syrinx with markers as imaged through the microscope. The left bronchus is removed to make the right MVM visible. Carbon micro-sphere markers 1–3 (red dots) represent the ML landmarks (see text). Abbreviations as in Fig. 1.
Figure 3
Figure 3
3D MVM morphology and tissue mass distribution. (A) Medio-lateral view of the zebra finch MVM (yellow) including cartilaginous pads (orange) and syringeal bones B1-B3 (grey). The thicker part that is referred to as the medial labium (ML) is outlined (black dotted line) based on sagittal sections in (BD) as indicated by the pink, blue and cyan outlines. The MVM is thicker in the roughly triangular shape between MVC, LDC and MDC cartilages embedded in the MVM. The slanted white dashed lines indicate where the cross-sectional areas of the ML were measured (see methods). (BD) Sagittal sections at locations shown in Panel A. The white arrowheads indicate the sudden narrowing that defines the border between ML and MTM. Abbreviations as in Fig. 1.
Figure 4
Figure 4
Typical raw MVC displacement and force data during a VS run. (A) Six cycles of MVC displacement (top) and resulting force (bottom) over time for the different experimental conditions; fixed cycles (blue lines), detached cycles (red), the difference force (black), and inertial forces of the setup (green) (see Methods). Each cycle starts with a positive displacement (defined as towards the motor, i.e. muscle shortening), pulling the MVC until peak displacement (here 0.86 mm) and back to resting position. The orange shaded area highlights the loading part of one cycle. (B) Force plotted against displacement resulting in work loops. Shown are 10 consecutive cycles (faded coloured lines) and the fitted loading curves (thick lines) for the three conditions. The loops run clockwise with the upper part of each curve representing the stretching or loading phase (solid arrows) and the lower part the unloading phase (faded arrows).
Figure 5
Figure 5
Stress-strain relationships for VS and MDS runs based on 3D stereoscopic marker tracking. (A) Inter-marker distances as engineering strain (v1, black; v2, red traces; left y-axis) and the displacement signal (light blue; right y-axis) during four successive cycles in a VS actuation run. At ~10% tissue strain, further displacement of MVC does not result in an increase in tissue strain. (BD) Marker positions (black dots) at (B) resting position of ML, (C) maximal strain of ML during VS actuation run and (D) maximal strain during MDS actuation run. The strain increase (red) in the direction of the muscle line of action (VS in C and MDS in D) is accompanied by a strain decrease (blue) in the perpendicular direction. The white dots in panel C indicate the resting position of the markers (panel B). The exact orientation of the x,y and z axes and reference point (0,0,0) are defined by the position of the calibration wand during the calibration procedure. Please see Supplementary Videos S1 and S2 online for animations of the strain changes in ML during a VS and MDS run.
Figure 6
Figure 6
Material properties of the medial labium in zebra finch based on syringeal muscle displacement. (A,B) Stress-strain relationship of the ML when lengthening the MVM in the (A) VS and (B) MDS runs (mean (black line) ± S.D. (green). (C,D) Elastic modulus as function of stress when lengthening the MVM in the (C) VS and (D) MDS runs (mean (black line) ± S.D. (blue). Mean, S.D. and range values of material properties and curve fitting parameters are listed in Table 1.
Figure 7
Figure 7
Predicted fundamental frequency range of zebra finch vocalizations based on string model. Fundamental frequency estimates (mean (black line) ± SD (green area)) for (A) VS and (B) MDS runs. The maximum strain achieved in the ML by modulating in situ muscle length is 11.9% and 3.9% for VS and MDS, respectively and indicated by red dots. Thus the predicted range of f 0 modulation is 350–787 Hz with VS and 392–497 Hz with MDS.

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