One-to-one innervation of vocal muscles allows precise control of birdsong

Abstract

The motor control resolution of any animal behavior is limited to the minimal force step available when activating muscles, which is set by the number and size distribution of motor units (MUs) and muscle-specific force. Birdsong is an excellent model system for understanding acquisition and maintenance of complex fine motor skills, but we know surprisingly little about how the motor pool controlling the syrinx is organized and how MU recruitment drives changes in vocal output. Here we developed an experimental paradigm to measure MU size distribution using spatiotemporal imaging of intracellular calcium concentration in cross-sections of living intact syrinx muscles. We combined these measurements with muscle stress and an in vitro syrinx preparation to determine the control resolution of fundamental frequency (f_o), a key vocal parameter, in zebra finches. We show that syringeal muscles have extremely small MUs, with 40%-50% innervating ≤3 and 13%-17% innervating a single muscle fiber. Combined with the lowest specific stress (5 mN/mm²) known to skeletal vertebrate muscle, small force steps by the major f_o controlling muscle provide control of 50-mHz to 7.3-Hz steps per MU. We show that the song system has the highest motor control resolution possible in the vertebrate nervous system and suggest this evolved due to strong selection on fine gradation of vocal output. Furthermore, we propose that high-resolution motor control was a key feature contributing to the radiation of songbirds that allowed diversification of song and speciation by vocal space expansion.

Keywords: birdsong; motor control; motor unit; songbird; sound production; vocal communication; voice.

Figure 1.. The songbird syrinx motor pool has very low mean innervation numbers.

A) Schematic of the motor control pathway of the songbird syrinx. B) Ventral view (left) and cross-section (right) of the syrinx with muscles highlighted in color. All muscle fibers run in parallel from rostral to caudal [22] as indicated by two white lines on the ventral view. C) Cross-sections through the tracheosyringeal branch of the hypoglossal nerve (NXIIts) and D) the syrinx (~800 stitched images) at mid-level, where all muscle fibers are present. The individual muscles are outlined in the same colors as in the cross-section in B. E) NXIIts axon and muscle fiber number and F) IN_mean per individual for a minimum estimate of 0 % (grey) and a maximum of 33 % (orange) sensory axons within NXIIts. See also Table S1. Data presented as mean ± SD. HVC: used as proper name, RA: robust nucleus of the arcopallium, nXIIts: tracheosyringeal portion of the hypoglossal nucleus, NXIIts: tracheosyringeal part of the hypoglossal nerve, DTB: musculus tracheobonchealis dorsalis, MDS: musculus syringealis dorsalis medialis, VS: musculus syringealis ventralis, VTB: musculus tracheobonchealis ventralis, * at p < 0.05.

Figure 2.. MU size distribution models predict one-to-one innervation in the songbird syrinx motor pool.

A) MU size distributions are skewed towards small units (bottom) and exponential in both anatomical connectome reconstructions and force-based measurements (top, and Figure S1). B) IN distribution as function of MU with IN_max ranging from 1 (gray) to 30 (blue). Black lines indicate the average IN_mean (horizontal dotted line) and distributed IN (stepped line) for the individual with the smallest IN_mean (3.84). The red line (also next panel) indicates the distribution for only superfast muscle fibers. See also Figure S2. C) The number of muscle fibers (horizontal lines) provides IN_max per individual. Colors correspond to IN_max lines in panel B. Shown are the individuals with smallest (3.84, gray) and largest IN_mean (5.67, blue). Shaded horizontal bars indicate IN range. D) Distribution and E) cumulative fraction of MU as a function of IN for superfast fibers with 0% (black line) or 33 % (orange line) adjustment for afferent sensory axons. Data presented as mean ± SD.

Figure 3.. The songbird syrinx motor pool has functional one-to-one innervation.

A) Schematic of setup to measure spatiotemporal resolved calcium spikes across the cross-section of an entire muscle. B) The muscle-nerve preparation (left) and fanned NXIIts nerve (right). C) Cross-section through the alive MDS muscle (3000 averages) showing all identified muscle fibers. D) Calcium dFF signal during field stimulation of the entire muscle shows alive (blue circles) and dead (red circles) muscle fibers. E) Example dFF frames during single motor nervlet stimulation of a motor unit with IN = 2 (left, muscle fibers 140 & 165) and IN = 1 (right, muscle fiber 129). F) Intensity color-coded raw dFF traces of all 172 muscle fibers during stepwise increase of the injected energy during 4 Hz stimulation by a single electrode. G) Raw dFF traces of 12 muscle fibers showing three MUs with IN = 1, 2 and 9, respectively. H) 10x averaged (top) and binary (bottom) dFF signal of active muscle fibers, sorted for onset timing showing two MUs with IN = 1 and two with IN = 2. I) Individual (top) and summed (bottom) IN distribution show that all individuals had several MUs that innervated a single muscle fiber (yellow bars). J) The summed distribution follows an exponential distribution (black line) that falls within the known MU distributions in other muscles [–29] (gray lines, Figure 2A, S1). See also Video S1.

Figure 4.. Songbird syringeal muscles generate the lowest stress.

Optimization of A) stimulus amplitude, B) frequency and C) muscle fiber length to determine P_o. D) Force profile after single (twitch) and tetanic (500 Hz) stimulation. Traces are the mean of eight twitch and three 500 Hz stimulations. E) P_o for syringeal muscles is 30 – 60x lower than skeletal (dark blue) muscles. F) P_o does not differ significantly between muscles (ANOVA: F = 0.24, df = 2 and 14, p = 0.79) G) Maximal force per muscle. H) Single fiber twitch force is not significantly different between muscles (ANOVA: F = 0.997, df = 2 and 14, p = 0.39)

Figure 5.. Syringeal MUs provide sub-Hertz resolution control of fundamental frequency.

A) Sound production paradigm in vitro actuating the insertion site of the f_o controlling ventral syringeal (VS) muscle. B) Example raw data of sound production induced by raising pressure (top), slow VS force modulation (middle) and resulting f_o changes (bottom). C) Frequency change as a function of VS force is linear over half the range of the average VS force modulation range of 0 – 5.31 mN in all 5 individuals (color coded). Short vertical lines on the x-axis indicate break point between linear and exponential curve fit for all individuals. See also Table S2. D) Distribution of the number of MUs within VS per force and f_o step available during maximal (tetanic) and E) minimal (twitch) force development.