Ultrasonic vocalization phenotypes in the Ts65Dn and Dp(16)1Yey mouse models of Down syndrome.

Down syndrome (DS) is a developmental disorder associated with a high incidence of challenges in vocal communication. DS can involve medical co-morbidities and structural social factors that may impact communication outcomes, which can present difficulties for the study of vocal communication challenges. Mouse models of DS may be used to study vocal communication differences associated with this syndrome and allow for greater control and consistency of environmental factors. Prior work has demonstrated differences in ultrasonic vocalization (USV) of the Ts65Dn mouse model of DS at a young adult age, however it is not known how USV characteristics are manifested at mature ages. Given that the aging process and age-related co-morbidities may also impact communication in DS, addressing this gap in knowledge may be of value for efforts to understand communication difficulties in DS across the lifespan. The current study hypothesized that the Ts65Dn and Dp(16)1Yey mouse models of DS would demonstrate differences in multiple measures of USV communication at a mature adult age of 5 months. METHODS: Ts65Dn mice (n = 16) and euploid controls (n = 19), as well as Dp(16)1Yey mice (n = 20) and wild-type controls (n = 22), were evaluated at 5 months of age for USV production using a mating paradigm. Video footage of USV sessions were analyzed to quantify social behaviors of male mice during USV testing sessions. USV recordings were analyzed using Deepsqueak software to identify 10 vocalization types, which were quantified for 11 acoustic measures. RESULTS: Ts65Dn, but not Dp(16)1Yey, showed significantly lower proportions of USVs classified as Step Up, Short, and Frequency Steps, and significantly higher proportions of USVs classified as Inverted U, than euploid controls. Both Ts65Dn and Dp(16)1Yey groups had significantly greater values for power and tonality for USVs than respective control groups. While Ts65Dn showed lower frequencies than controls, Dp(16)1Yey showed higher frequencies than controls. Finally, Ts65Dn showed reductions in a measure of complexity for some call types. No significant differences between genotype groups were identified in analysis of behaviors during testing sessions. CONCLUSION: While both Ts65Dn and Dp(16)1Yey show significant differences in USV measures at 5 months of age, of the two models, Ts65Dn shows a relatively greater numbers of differences. Characterization of communication phenotypes in mouse models of DS may be helpful in laying the foundation for future translational advances in the area of communication difficulties associated with DS.

Effects of social isolation on 50-kHz ultrasonic vocalizations, affective state, cognition, and neurotransmitter concentrations in the ventral tegmental and locus coeruleus of adult rats.

Vocal communication, cognition, and affective state are key features of sustained health and wellness, and because vocalizations are often socially-motivated, social experience likely plays a role in these behaviors. The monoaminergic systems of the ventral tegmental area (VTA) and the locus coeruleus (LC) are associated with social and reward processing, vocalization production, and neurotransmitter changes in response to environmental stressors. The effect of social isolation on these complex behaviors and the underlying neural mechanisms is relatively unknown. To add to this body of literature, we randomized adult male Long-Evans rats to control (housed with a cagemate) or isolated (housed individually) conditions and assayed ultrasonic vocalizations, cognition (novel object recognition test), anxiety (elevated plus maze) and anhedonia (sucrose preference test) at 2, 4, 6, 8, and 10 months of age. At 10 months, VTA and LC samples were assayed for dopamine, norepinephrine, and serotonin using high performance liquid chromatography. We tested the hypotheses that isolation 1) diminishes vocalizations and cognition, 2) increases anxiety and depression, and 3) increases levels of dopamine, norepinephrine, and serotonin in the VTA and LC. Results showed isolation significantly reduced vocalization tonality (signal-to-noise ratio) and increased maximum frequency. There were no significant findings for cognition, anxiety, or anhedonia. Dopamine and serotonin and their respective metabolites were significantly increased in the VTA in isolated rats. These findings suggest chronic changes to social condition such as isolation affects vocalization production and levels of VTA neurotransmitters.

Changes in ultrasonic vocalizations after unilateral cerebral ischemia in a rat stroke model.

Stroke frequently results in communication impairments that negatively impact quality of life and overall recovery, yet the biological mechanisms underlying these changes are not well understood. Ultrasonic vocalizations (USVs) in rodent models of disease and aging have been used to improve our understanding of the biological mechanisms that underlie vocal deficits and their response to interventions. Changes in USVs after middle cerebral artery occlusion (MCAO) in mice have been reported, yet rat models have significant anatomical and behavioral advantages over mice, including the ability to vocally train rats with an established paradigm. We sought to determine whether a unilateral MCAO rat stroke model provides a biologically and behaviorally relevant way to study post stroke vocalization deficits. We hypothesized that left MCAO would be associated with changes in USVs. Six weeks after MCAO or sham-control surgery, USVs were recorded in rats using an established mating paradigm. Stroke was associated with differences in USV acoustics including more frequent use of simple calls characterized by shorter durations and restricted bandwidths. These parameters were also found to correlate with post stroke lingual weakness. This is the first study to describe changes to rat USVs using a stroke model. These results suggest the unilateral MCAO rat stroke model is a biologically and behaviorally relevant model to understand how stroke affects vocal behaviors.

Semi-Automated Training of Rat Ultrasonic Vocalizations.

Rats produce ultrasonic vocalizations (USVs) for conspecific communication. These USVs are valuable biomarkers for studying behavioral and mechanistic changes in a variety of diseases and disorders. Previous work has demonstrated operant conditioning can progressively increase the number of USVs produced by rats over multiple weeks. This operant conditioning paradigm is a useful model for investigating the effects of increased laryngeal muscle use on USV acoustic characteristics and underlying central and peripheral laryngeal sensorimotor mechanisms. Previous USV operant conditioning studies relied on manual training to elicit USV productions, which is both time and labor intensive and can introduce human variability. This manuscript introduces a semi-automated method for training rats to increase their rate of USV production by pairing commercially available operant conditioning equipment with an ultrasonic detection system. USV training requires three basic components: elicitation cue, detection of the behavior, and a reward to reinforce the desired behavior. With the semi-automated training paradigm, indirect exposure to the opposite sex or an olfactory cue can be used to elicit USV production. The elicited USV is then automatically detected by the ultrasonic acoustic system, which consequently triggers the release of a sucrose pellet reward. Our results demonstrate this semi-automated procedure produces a similar increase in USV production as the manual training method. Through automation of USV detection and reward administration, staffing requirements, human error, and subject behavioral variability may be minimized while scalability and reproducibility are increased. This automation may also result in greater experimental flexibility, allowing USV training paradigms to become more customizable for a wider array of applications. This semi-automated USV behavioral training paradigm improves upon manual training techniques by increasing the ease, speed, and quality of data collection.

The Relationship Between Menopause and Dysphagia: A Scoping Review.

Purpose: Menopause marks the end of fertility and rapid decline of ovarian hormones in the female body, which corresponds to a myriad of changes to bodily systems, including the upper aerodigestive tract. Despite substantial evidence that menopause negatively impacts oral health, bones, and skeletal muscles, little research has examined these effects as they relate to swallowing. The purpose of this scoping review was to compile and summarize the existing literature investigating the relationship between menopause and swallowing-related structures and physiology. Methods: Search terms were selected for three databases (PubMed, Scopus, and CINAHL) to gather relevant literature evaluating the relationship between menopause and swallowing-related anatomy as well as swallowing functions in both human and animal models. Relevant articles were reviewed, collated, and summarized to synthesize findings, identify gaps in the literature, and provide suggestions for future directions. Results: This scoping review yielded 204 studies with the majority of these studies relating to one or more of the following categories: oral health, saliva, mandibular structures, and taste. Common oral symptoms reported in the literature included xerostomia, hyposalivation, tooth decay, inflammation of oral mucosa, and oral pain. Although literature supports that menopause adversely affects oral health, saliva, mandibular structures, and alters taste, a dearth of information was evident regarding how these hormone-dependent changes can adversely affect swallowing. Conclusions: The relationship between menopause and swallowing has been overlooked by field of speech-language pathology. By identifying the major gaps in the literature, these results will inform future investigations evaluating relationships among ovarian hormones and swallowing.

Biological and Acoustic Sex Differences in Rat Ultrasonic Vocalization.

The rat model is a useful tool for understanding peripheral and central mechanisms of laryngeal biology. Rats produce ultrasonic vocalizations (USVs) that have communicative intent and are altered by experimental conditions such as social environment, stress, diet, drugs, age, and neurological diseases, validating the rat model's utility for studying communication and related deficits. Sex differences are apparent in both the rat larynx and USV acoustics and are differentially affected by experimental conditions. Therefore, the purpose of this review paper is to highlight the known sex differences in rat USV production, acoustics, and laryngeal biology detailed in the literature across the lifespan.

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

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.

The Effects of Menopause on Neuromuscular Parameters of the Rat Vocal Folds.

OBJECTIVES/HYPOTHESIS: Menopause adversely affecs power and endurance of the limb muscles. However, despite clinical observations that menopause corresponds to negative changes of the voice, the direct effects of estrogen deprivation on the thyroarytenoid muscles are unknown. The purpose of this study was to investigate the effects of estrogen deprivation via ovariectomy on three neuromuscular parameters of the thyroarytenoid muscles using a rat model. STUDY DESIGN: Animal model. METHODS: Cryosections of vocal folds of 20 (10 control and 10 ovariectomized) female rats were stained to label neuromuscular junctions, fiber size, or parvalbumin levels using immunohistochemical techniques and compared between experimental groups. RESULTS: The neuromuscular junctions, thyroarytenoid fiber sizes, and parvalbumin levels of the vocal folds were similar between experimental groups. CONCLUSIONS: The loss of estrogen did not change neuromuscular parameters of the vocal folds of adult female rats; therefore, vocal changes within the outer vibratory layers of the vocal folds may primarily be responsible for clinically observed menopausal vocal changes. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1343-1348, 2021.

Identifying Concomitant Health Conditions in Individuals With Chronic Voice Problems.

OBJECTIVE: Current clinical practice guidelines recommend a laryngoscopic referral for patients who present with hoarseness for longer than 28 days and earlier for patients with certain high-risk factors. The goal of this study was to identify additional possible concomitant health conditions in individuals with chronic (>28 days) hoarseness to improve early detection of chronic voice problems. METHODS: Using data from the 2012 National Health Interview Survey, four health conditions were selected: Swallowing problems, respiratory problems, hormonal-cycle problems, and physical activity limitations. Multivariable logistic regressions controlling for age, gender, race, and smoking status, were used to calculate the odds ratios for the association of each of these four health conditions to chronic versus acute voice problems. RESULTS: Of the 2,746 respondents who reported a voice disorder within the last year, 736 reported a voice problem lasting longer than 4 weeks in duration. After controlling for covariates, individuals reporting swallowing problems and physical activity limitations were more likely to report a chronic voice problem versus an acute voice problem, odds ratios with 95% confidence intervals of 1.983 (1.619, 2.430) and 1.716 (1.355, 2.173), respectively. No significant associations were found for respiratory or hormonal-cycle problems. CONCLUSION: Individuals who present with both a voice problem and a swallowing problem or physical activity limitation may be at increased risk for developing a chronic voice problem. Therefore, these two health conditions should be included as high-risk factors when determining the escalation of care for a patient presenting with acute dysphonia.

The effects of the estrous cycle, menopause, and recording condition on female rat ultrasonic vocalizations.

The goal of this study was to evaluate the effects of ovarian hormones on female rat ultrasonic vocalizations (USVs). Twenty (10 control and 10 ovariectomized) 3-month-old female rats were recorded in 3 recording conditions (elicitation, dyad, and isolation) over a full estrous cycle or time-matched duration. There were differences in USV acoustics (frequency and complexity parameters) across recording conditions but no differences in USV acoustics between control and ovariectomized groups. USVs produced in isolation had lower frequency and complexity parameters than elicited USVs for both control and ovariectomized rats. Additionally, for control rats, USV parameters of frequency, complexity, duration, and intensity changed depending on the estrous state. Therefore, although fluctuating hormone levels may influence USV acoustics, this variation can be controlled for by ovariectomizing female rats.

Correction to: Volumetric Changes to the Pharynx in Healthy Aging: Consequence for Pharyngeal Swallow Mechanics and Function.

This erratum notifies the readers of the Dysphagia journal of an error in the original published version of this manuscript. In that manuscript, a previously available open source spreadsheet tool was used to calculate the position of the posterior laryngeal air column on lateral view videofluoroscopic images as a proxy for the bottom of the pharynx. We have subsequently been made aware of an error in the mathematical formula built into the spreadsheet, which resulted in a reversal of the results for the X and Y planes of measurement. This erratum provides corrections to the results and interpretations of the original manuscript.

Preparation of the Rat Vocal Fold for Neuromuscular Analyses.

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

Variations in Healthy Swallowing Mechanics During Various Bolus Conditions Using Computational Analysis of Swallowing Mechanics (CASM).

Bolus properties such as volume, consistency, and density have been shown to influence swallowing through the analysis of kinematics and timing in both normal and disordered swallowing. However, inherent intra- and inter-person variability of swallowing cloud interpretation of group data. Computational analysis of swallow mechanics (CASM) is an established methodology that uses coordinate tracking to map structural movements during swallowing and yields statistically powerful analyses at both the group and individual levels. In this study, the CASM method was used to determine how different bolus properties (volume, consistency, and density) altered swallow mechanics in healthy young adults at the group and individual levels. Videofluoroscopic swallow studies of 10 (4 females) healthy young adults were analyzed using CASM. Five bolus types were administered in each study (3 × 5 ml 40% w/v nectar, 3 × 5 ml 22% w/v thin, 3 × 5 ml 40% w/v thin, 3 × 10 ml 22% w/v thin, and 3 × 20 ml 22% w/v thin). Canonical variate analyses demonstrated that bolus condition did not affect swallowing mechanics at the group level, but bolus condition did affect pharyngeal swallow mechanics at the individual level. Functional swallow adaptations (e.g., hyoid movement) to bolus conditions were not uniform across participants, consistent with the nonsignificant group finding. These results suggest that individual swallowing systems of healthy young individuals vary in how they respond to bolus different conditions, highlighting the intrinsic variability of the swallow mechanism and the importance of individually tailored evaluation and treatment of swallowing. Findings warrant further investigation with different bolus conditions and aging and disordered populations.

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

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.

A Tutorial of the Effects of Sex Hormones on Laryngeal Senescence and Neuromuscular Response to Exercise.

Purpose The purpose of this tutorial is to summarize how sex hormones affect both laryngeal senescence and neuromuscular response to exercise, highlighting the importance of considering sex differences in developing treatment for the senescent voice. Conclusion Men and women's voices are sexually dimorphic throughout the life span, including during the laryngeal adaptations observed during senescence. Therefore, presbyphonia (age-related dysphonia) likely clinically manifests differently for men and women due to differences in how the male and the female larynx change in response to aging. Because sexual dimorphism is evident in both laryngeal aging and response to exercise, voice therapy programs aimed at treating the typical and disordered aged voice should consider sex differences in their design.

Volumetric Changes to the Pharynx in Healthy Aging: Consequence for Pharyngeal Swallow Mechanics and Function.

Pharyngeal lumen volume is prone to increase as a consequence of pharyngeal muscle atrophy in aging. Yet, the impact of this on swallowing mechanics and function is poorly understood. We examined the relationship between pharyngeal volume and pharyngeal swallowing mechanics and function in a sample of healthy community-dwelling seniors. Data were collected from 44 healthy seniors (21 male, mean age = 76.9, SD = 7.1). Each participant swallowed 9 boluses of barium (3 × 5 ml thin, 3 × 20 ml thin, 3 × 5 ml nectar). Pharyngeal shortening, pharyngeal constriction, pyriform sinus and vallecular residue were quantified from lateral view videofluorosopic swallowing studies. Pharyngeal lumen volume was captured during an oral breathing task with acoustic pharyngometry. In addition, within-participant measures of strength and anthropometrics were collected. Four linear mixed effects regression models were run to study the relationship between pharyngeal volume and pharyngeal constriction, pharyngeal shortening, pyriform sinus residue, and vallecular residue while controlling for bolus condition, age, sex, and posterior tongue strength. Increasing pharyngeal lumen volume was significantly related to worse constriction and vallecular residue. In general, larger and thicker boluses resulted in worse pharyngeal constriction and residue. Pharyngeal shortening was only significantly related to posterior tongue strength. Our work establishes the utility of acoustic pharyngometry to monitor pharyngeal lumen volume. Increasing pharyngeal lumen volume appears to impact both pharyngeal swallowing mechanics and function in a sample of healthy, functional seniors.

Sexual dimorphism in laryngeal muscle fibers and ultrasonic vocalizations in the adult rat.

OBJECTIVE: The human voice is sexually dimorphic in obvious ways, such as differences in fundamental frequency and gross laryngeal anatomy, but also in less apparent ways, such as in the prevalence and types of voice disorders and the manifestation of voice changes in advanced age. Differences between males and females are rarely explored, however, in mechanistic animal studies. The goal of this study was to explore sexual dimorphism in laryngeal function and structure in adult rats by examining ultrasonic vocalization acoustics and muscle fiber size and type in the thyroarytenoid muscle. STUDY DESIGN: Animal group comparison. METHODS: Spontaneous ultrasonic vocalizations from 10 male adult rats and 10 female adult rats were recorded, classified, and acoustically analyzed. Cross-sections of the thyroarytenoid muscle were stained and imaged for analysis of muscle fiber size and type. Acoustic and muscle parameters were statistically compared between sexes. RESULTS: Male rats had a lower mean frequency of short ultrasonic vocalizations. Male rats also had a larger mean fiber size in the external division of the thyroarytenoid and larger overall muscle area in both the vocalis and external divisions of the thyroarytenoid. However, muscle fiber type compositions were similar between sexes in both the vocalis and external division of the thyroarytenoid muscles. CONCLUSION: Functional and structural laryngeal differences exist between adult male and female rats; therefore, the rat model can be used to further study sexual dimorphism of the voice. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E270-E276, 2017.