Male and female syringeal muscles exhibit superfast shortening velocities in zebra finches

Abstract

Vocalisations play a key role in the communication behaviour of many vertebrates. Vocal production requires extremely precise motor control, which is executed by superfast vocal muscles that can operate at cycle frequencies over 100 Hz and up to 250 Hz. The mechanical performance of these muscles has been quantified with isometric performance and the workloop technique, but owing to methodological limitations we lack a key muscle property characterising muscle performance, the force-velocity relationship. Here, we quantified the force-velocity relationship in zebra finch superfast syringeal muscles using the isovelocity technique and tested whether the maximal shortening velocity is different between males and females. We show that syringeal muscles exhibit high maximal shortening velocities of 25L0 s-1 at 30°C. Using Q10-based extrapolation, we estimate they can reach 37-42L0 s-1 on average at body temperature, exceeding other vocal and non-avian skeletal muscles. The increased speed does not adequately compensate for reduced force, which results in low power output. This further highlights the importance of high-frequency operation in these muscles. Furthermore, we show that isometric properties positively correlate with maximal shortening velocities. Although male and female muscles differ in isometric force development rates, maximal shortening velocity is not sex dependent. We also show that cyclical methods to measure force-length properties used in laryngeal studies give the same result as conventional stepwise methodologies, suggesting either approach is appropriate. We argue that vocal behaviour may be affected by the high thermal dependence of superfast vocal muscle performance.

Keywords: V max; Isovelocity; Muscle performance; Song learning; Vocal performance.

Fig. 1.

Force–velocity protocol and analysis example. (A) Length changes at different ramp velocities. (B) Total muscular stress when stimulated, where the muscle tetanised before being shortened at the differing ramp velocities. (C) Passive stress during shortening. (D) Active stress, calculated as total stress (B) minus passive stress (C). Insets in A–D show a zoomed version of the ramp process. Graded shades of grey are used to represent different ramp velocities. (E) Resulting force–velocity profile including the data shown in A–D using points of corresponding colours; green points show data not included in A–D. (F) Power output derived from the force–velocity profile; powers calculated from shown data are displayed using points of corresponding colours to lines shown in A–D; green points indicate data not shown. Purple lines in E and F are used to highlight the maximum shortening velocity (V_max), stress at peak power (σ at Π_i), velocity at peak power (V at Π_i) and peak power (Π_i). Data shown are left DTB preparation from female GW423 at 30°C using velocity-capped protocol 1 (no-step).

Fig. 2.

Cyclic and stepwise protocols provide the same optimal length and stress in vocal muscles. (A) Stepwise approach muscle length is increased in steps and tetanised at each length. (B) Resulting profile from this approach with active force shown in red and passive in black, vertical dashed line shows the optimal length (L₀). (C) Cyclic approach, muscle is stimulated throughout a 1 Hz length change (±0.5 mm from starting length), top panel shows length change, and bottom shows resulting stress outputs. Black line shows the passive stress, purple total stress and red active stress (calculated as total stress minus passive stress). (D) Resulting profile from approach in C; active stress shown in red and passive in black, the vertical dashed line indicates L₀. Yellow boxes in A and C are used to show the stimulation period. (E) L₀ from the stepwise and cyclic methodologies. Solid filled orange squares (male; N=8) and grey circles (female; N=6) show the mean±s.d., translucent squares and circles show raw data. (F) The stress (σ₀) at the optimal length during stepwise and cyclic methodologies. Solid filled orange squares (male) and grey circles (female) show the mean±s.d., translucent squares and circles show raw data. (G) Isometric stress before and after being stimulated for 1 s. Stress 5 min after prolonged stimulation did not significantly differ from previous tetani. Male data (N=8) are shown in orange, and female (N=6) in grey, solid colours show the mean±s.d., translucent show individual data points.

Fig. 3.

Force–velocity and force–power relationships in zebra finch syrinx muscle. (A,C) Force–velocity curves of male (orange; N=8) dorsal tracheobronchial (DTB) muscle at (A) 20°C and (C) 30°C. (B,D) Force–velocity curves of female (grey; N=9) DTB muscle at (B) 20°C and (D) 30°C. Insets show the resulting force–power relationships, calculated as force×velocity. The mean (solid) and standard deviation (dashed) are thick lines, whereas individual curves are translucent. All shown data was obtained using velocity-capped protocol 1 (no-step).

Fig. 4.

Maximum shortening velocity extrapolation of DTB muscle to body temperature. (A) Shortening velocity at 40°C estimated using Q₁₀ values calculated from measurements at 20 and 30°C in male (orange; N=8) and female (grey; N=9) DTB muscle, and (B) assuming a conservative Q₁₀ value of 1.50. Means±s.d. are shown as filled squares and circles; raw data are shown as translucent. The mean±s.d. Q₁₀ temperature coefficients are shown for each sex. Male and female data are slightly offset in temperature for clarity. All shown data were obtained using velocity-capped protocol 1 (no-step).

Fig. 5.

Isometric properties that predict muscle shortening velocity. Isometric contractile features of DTB muscle at 30°C: (A) time to peak twitch (t_Ptw), (B) full width at half maximal force time (FWHM), (C) twitch half relaxation time (RT₅₀) and (D) time to peak tetanus (t_P0). Insets in A–D give examples of how these parameters were measured. (E–H) The relationship between V_max and (E) t_Ptw, (F) FWHM, (G) RT₅₀ and (H) t_P0. Correlation equations shown in E, F and H were all significant, with respective P-values of 0.045, 0.048 and 0.018. Data from males (N=8) are shown in orange, and females (N=9) in grey, translucent colours show individual datapoints, solid colours show the means±s.d., asterisks indicate significant differences (*P≤0.05 and ***P≤0.001). All V_max values were obtained using velocity-capped protocol 1 (no-step).