Laryngeal Neuromuscular Response to Short- and Long-Term Vocalization Training in Young Male Rats

Abstract

Purpose Although vocal training is often purported to restore and rebalance laryngeal muscle function, little is known about the direct effects of vocal training on the laryngeal muscles themselves. Consequently, parameters of vocal exercise dose, such as training duration and intensity, have not been well defined. The goal of this study was to use a behavioral animal model to determine the effects of short- and long-term ultrasonic vocalization (USV) training on USV acoustics, thyroarytenoid (TA) muscle neuromuscular junctions (NMJs), and TA muscle fiber size in adult rats. Method Twenty-four young adult male Long-Evans rats were divided into 3 groups (untrained control, 4-week training, and 8-week training). Baseline and posttraining USVs were recorded and acoustically analyzed for fundamental frequency, frequency bandwidth, amplitude, and duration. Presynaptic and postsynaptic NMJ morphological features and muscle fiber size were measured in the TA. Results USV training had no effect on USV acoustics. Eight weeks of USV training, however, resulted in a lower NMJ motor endplate dispersion ratio, consistent with previous findings. USV training did not affect fiber size within the TA muscle. Conclusions This study demonstrated that 8 weeks of USV training can induce peripheral neural adaptations in the NMJ of the TA muscle in young rats. The observed adaptations suggest that vocal training is consistent with endurance-type exercise, but the adaptations occur on a longer time scale than similar adaptations in the limb muscles.

Figure 1.

Muscle fibers outlined in blue in the medial thyroarytenoid and lateral thyroarytenoid of the vocal fold at 20× magnification. Type IIx muscle fibers were stained red in the vocal fold to distinguish the medial thyroarytenoid, which is primarily composed of Type IIx muscle fibers, from the lateral thyroarytenoid, which has no Type IIx muscle fibers.

Figure 2.

Representative neuromuscular junction images from control (A), 4-week (B), and 8-week (C) animals after automatic image processing and rotation to a standardized en face position. The presynaptic nerve terminal is pseudocolored green, the postsynaptic motor endplate is pseudocolored red, and the overlap between the two is indicated in yellow. The scale bar in each panel is equal to 10 µm.

Figure 3.

The mean and standard error of the number of vocalizations produced each week during each vocal training session (4- and 8-week groups) or the control interactions (control group). The trajectory of the weekly increase was the same for the two trained groups, although the individual variation in the number of ultrasonic vocalizations (USVs) produced increased each successive week. The control group interactions elicited few to no USVs.

Figure 4.

There were no interaction effects between training group (8-week, 4-week, and control) and time point (baseline or postexperiment) in any of the four ultrasonic vocalization acoustic measures, indicating no effect of training on ultrasonic vocalization acoustics. Data are shown as mean and standard error. Amplitude was measured in decibels (full scale), meaning all recorded amplitudes were negative relative to the maximum amplitude of the recording system, which was defined as 0 dB.

Figure 5.

There were no differences in muscle fiber size between training groups within the lateral thyroarytenoid (LTA) muscle or the medial thyroarytenoid (MTA) muscle. Data are shown as mean and standard error.

Figure 6.

Plot of the regression analysis between maximum ultrasonic vocalization amplitude at postexperiment recordings and neuromuscular junction motor endplate dispersion ratio in the thyroarytenoid muscle. The model demonstrated a significant negative relationship, F(1, 18) = 6.52, p = .02, with an R ² of 26%. Each point represents data from one animal (x = untrained control group, open circle = 4-week trained group, filled circle = 8-week trained group). USV = ultrasonic vocalization.