Effect of aging on ultrasonic vocalizations and laryngeal sensorimotor neurons in rats

Abstract

While decline in vocal quality is prevalent in an aging population, the underlying neurobiological mechanisms contributing to age-related dysphonia are unknown and difficult to study in humans. Development of an animal model appears critical for investigating this issue. Using an established aging rat model, we evaluated if 50-kHz ultrasonic vocalizations in 10, 32-month-old (old) Fischer 344/Brown Norway rats differed from those in 10, 9-month-old (young adult) rats. The retrograde tracer, Cholera Toxin β, was injected to the thyroarytenoid muscle to determine if motoneuron loss in the nucleus ambiguus was associated with age. Results indicated that older rats had vocalizations with diminished acoustic complexity as demonstrated by reduced bandwidth, intensity, and peak frequency, and these changes were dependent on the type of 50-kHz vocalization. Simple calls of old rats had reduced bandwidth, peak frequency, and intensity while frequency-modulated calls of old rats had reduced bandwidth and intensity. Surprisingly, one call type, step calls, had increased duration in the aged rats. These findings reflect phonatory changes observed in older humans. We also found significant motoneuron loss in the nucleus ambiguus of aged rats, which suggests that motoneuron loss may be a contributing factor to decreased complexity and quality of ultrasonic vocalizations. These findings suggest that a rat ultrasonic phonation model may be useful for studying age-related changes in vocalization observed in humans.

Fig. 1

Ultrasonic vocalization call types. Representative calls from young (9 month) and old (32 month) rats are shown. The three call-type categories include simple (constant frequency), step (varying frequencies, generally slow to change between frequencies), and frequency-modulated, or FM (trill-like). Relative intensity is represented with color, as depicted in the relative intensity scale shown

Fig. 2

Immunohistochemistry of retro-labeled motoneurons in the compact formation of the nucleus ambiguus. a Motoneurons stained for immunoreactivity to the retrograde tracer, Cholera Toxin β. b Mature neurons stained for immunoreactivity to the mature neuronal marker, NeuN. c An overlay of the two antibodies. d View of thyroarytenoid muscles of a F344/Brown Norway rat provided by the microlaryngoscope and microscope (magnification of × 32 with a × 1.6 lenspiece)

Fig. 3

Duration of calls. Means and standard error of the mean (SEM) of the average duration of simple, step, and frequency-modulated calls of young (n = 10) and old animals (n = 8). *p < 0.05

Fig. 4

a Average Bandwidth. Means and standard error of the mean (SEM) of the average bandwidth of simple, step, and frequency-modulated calls of young (n = 10) and old animals (n = 8). b Maximum Bandwidth. Means and standard error of the mean (SEM) of the maximum peak frequency of simple, step, and frequency-modulated calls of young (n = 10) and old animals (n = 8). *p < 0.05

Fig. 5

a Average Peak Frequency. Means and standard error of the mean (SEM) of the average peak frequency of simple, step, and frequency-modulated calls of young (n = 10) and old animals (n = 8). b Maximum Peak Frequency. Means and standard error of the mean (SEM) of the maximum peak frequency of simple, step, and frequency-modulated calls of young (n = 10) and old animals (n = 8). *p < 0.05

Fig. 6

a Average Intensity. Means and standard error of the mean (SEM) of the average intensity of simple, step, and frequency-modulated calls of young (n = 10) and old rats (n = 8). b Maximum intensity. Means and standard error of the mean (SEM) of the maximum intensity of simple, step, and frequency-modulated calls of young (n = 10) and old rats (n = 8). Please note that intensity, measured in decibels (dB), is a negative value, and a less negative value reflects a louder vocalization. *p < 0.05

Fig. 7

Motoneuron Number. Mean and standard error of the mean (SEM) of the number of motoneurons retro-traced with CTβ from the thyroarytenoid muscle in the larynx to the nucleus ambiguus of young (n = 8) and old animals (n = 8). *p < 0.05