Effects of Vocal Training on Thyroarytenoid Muscle Neuromuscular Junctions and Myofibers in Young and Older Rats

Abstract

The purpose of this investigation was to determine the effects of vocal training on neuromuscular junction (NMJ) morphology and muscle fiber size and composition in the thyroarytenoid muscle, the primary muscle in the vocal fold, in younger (9-month) and older (24-month) Fischer 344 × Brown Norway male rats. Over 4 or 8 weeks of vocal training, rats of both ages progressively increased their daily number of ultrasonic vocalizations (USVs) through operant conditioning and were then compared to an untrained control group. Neuromuscular junction morphology and myofiber size and composition were measured from the thyroarytenoid muscle. Acoustic analysis of USVs before and after training quantified the functional effect of training. Both 4- and 8-week training resulted in less NMJ motor endplate dispersion in the lateral portion of the thyroarytenoid muscle in rats of both ages. Vocal training and age had no significant effects on laryngeal myofiber size or type. Vocal training resulted in a greater number of USVs with longer duration and increased intensity. This study demonstrated that vocal training induces laryngeal NMJ morphology and acoustic changes. The lack of significant effects of vocal training on muscle fiber type and size suggests vocal training significantly improves neuromuscular efficiency but does not significantly influence muscle strength changes.

Keywords: Acoustics; Laryngeal muscle; Neuromuscular junction; Presbyphonia; Vocal fold atrophy.

Figure 1.

(A) Motor endplate dispersion ratio was smaller in both the 4- and 8-week training groups in both younger and older animals in the lateral thyroarytenoid (LTA) muscle, but not the medial thyroarytenoid (MTA) muscle. Representative images of motor endplates from animals in the (B) trained and (C) untrained control groups. Significant differences within each age group are indicated in Figure 1A as follows: * = experimental group significantly different from control group (age groups combined). Dark dots in the box and whisker plots represent the median and the box the interquartile range; the whiskers extend to the 95% confidence interval for the median. Circles outside the boxes represent outliers. Scale bar = 5 µm.

Figure 2.

Representative images of poor synaptic overlap (left) and robust overlap (right) of the motor endplate (red) and nerve terminal (green). Overlap is indicated by yellow. (color online) Scale bar = 5 µm.

Figure 3.

Average minimum Feret diameter muscle fiber size for lateral thyroarytenoid (LTA), medial thyroarytenoid (MTA), and plantaris hindlimb. Although there were average increases in the LTA of young animals with training and average decreases in the plantaris hindlimb of young with training, these training differences were not statistically significant. Any differences in responses between muscle type could have to do with individual variability across groups or due to task specificity, where the larynx was exercised more than the limbs. Training effects seemed to have even less impact on the laryngeal and plantaris hindlimb muscles in the older cohort. Box and whisker plots are expressed as medians, interquartile ranges, and outliers.

Figure 4.

Change in ultrasonic vocalization intensity (delta power in dB/kHz) from baseline to post-experiment by experimental group and age. Significant differences within each age group are indicated as follows: * = different than control group; # = different from 4-week group.