Relationships Between Vocal Fold Adduction Patterns, Vocal Acoustic Quality, and Vocal Effort in Individuals With and Without Hyperfunctional Voice Disorders.

OBJECTIVES/HYPOTHESIS: Increased vocal effort and aberrant vocal quality are often attributed to vocal fold hyperadduction in hyperfunctional voice disorders. However, there are currently no established methods to quantify vocal fold adduction beyond subjective descriptors in this clinical population. Furthermore, relationships between vocal fold adduction patterns, vocal effort severity, and vocal quality are not well characterized. Therefore, the objectives of this study were to (1) quantify vocal fold adduction, applying a previously validated method developed for patients with vocal fold paralysis, and (2) correlate these measures with acoustic vocal quality and self-perceived measures of vocal effort severity. METHODS: A deep learning program, Automated Glottic Action Tracking using artificial Intelligence, was used to track glottic angle configurations and vocal fold adduction velocities on laryngoscopic videos across 60 laryngoscopies (20 primary muscle tension dysphonia [pMTD], 20 phonotraumatic lesions, and 20 healthy controls). Voice samples were also acquired, and cepstral peak prominence (CPP) and H1-H2 acoustic measures were used to quantify vocal quality. Participants were also asked to rate their vocal effort on a 100 mm visual analog scale. RESULTS: There were no significant group differences in glottic angle configurations or vocal fold adduction velocities, although there were trends toward increased peak vocal fold adduction velocities in patients with hyperfunctional voice disorders compared to controls. Vocal effort was significantly higher in the two hyperfunctional groups compared to controls. CPP was significantly lower in the pMTD group, but there were no group differences in acoustic parameters between any of the other groups or for H1-H2 values. CONCLUSION: Despite significantly more vocal effort reported in patients with hyperfunctional voice disorders, there were no significant group differences in vocal fold adduction patterns. These findings suggest other physiologic mechanisms may also be responsible for the symptoms and genesis of pMTD and benign vocal fold lesions.

Optical Flow Analysis of Paralaryngeal Muscle Movement.

OBJECTIVES: The paralaryngeal muscles are thought to be hyperfunctional with phonation in patients with primary muscle tension dysphonia (pMTD). However, objective, quantitative tools to assess paralaryngeal movement patterns lack. The objectives of this study were to (1) validate the use of optical flow to characterize paralaryngeal movement patterns with phonation, (2) characterize phonatory optical flow velocities and variability of the paralaryngeal muscles before and after a vocal load challenge, and (3) compare phonatory optical flow measures to standard laryngoscopic, acoustic, and self-perceptual assessments. METHODS: Phonatory movement velocities and variability of the paralaryngeal muscles at vocal onsets and offsets were quantified from ultrasound videos and optical flow methods across 42 subjects with and without a diagnosis of pMTD, before and after a vocal load challenge. Severity of laryngoscopic mediolateral supraglottic compression, acoustic perturbation, and ratings of vocal effort and discomfort were also obtained at both time points. RESULTS: There were no significant differences in optical flow measures of the paralaryngeal muscles with phonation between patients with pMTD and controls. Patients with pMTD had significantly more supraglottic compression, higher acoustic perturbations, and higher vocal effort and vocal tract discomfort ratings. Vocal load had a significant effect on vocal effort and discomfort but not on supraglottic compression, acoustics, or optical flow measures of the paralaryngeal muscles. CONCLUSION: Optical flow methods can be used to study paralaryngeal muscle movement velocity and variability patterns during vocal productions, although the role of the paralaryngeal in pMTD diagnostics (e.g., vocal hyperfunction) remains suspect. LEVEL OF EVIDENCE: 2 Laryngoscope, 134:1792-1801, 2024.

Acoustic and Physiologic Correlates of Vocal Effort in Individuals With and Without Primary Muscle Tension Dysphonia.

OBJECTIVES: The aims of this study were to determine relationships between vocal effort and (a) acoustic correlates of vocal output and (b) supraglottic compression in individuals with primary muscle tension dysphonia (pMTD) and without voice disorders (controls) in the context of a vocal load challenge. METHOD: Twenty-six individuals with pMTD and 35 vocally healthy controls participated in a 30-min vocal load challenge. The pre- and postload relationships among self-ratings of vocal effort, various acoustic voice measures, and supraglottic compression (mediolateral and anteroposterior) were tested with multiple regression models and post hoc Pearson's correlations. Acoustic measures included cepstral peak prominence (CPP), low-to-high spectral ratio, difference in intensity between the first two harmonics, fundamental frequency, and sound pressure level (dB SPL). RESULTS: Regression models for CPP and mediolateral compression were statistically significant. Vocal effort, diagnosis of pMTD, and vocal demand were each significant variables influencing CPP measures. CPP was lower in the pMTD group across stages. There was no statistical change in CPP following the vocal load challenge within either group, but both groups had an increase in vocal effort postload. Vocal effort and diagnosis influenced the mediolateral compression model. Mediolateral compression was higher in the pMTD group across stages and had a negative relationship with vocal effort, but it did not differ after vocal loading. CONCLUSIONS: CPP and mediolateral supraglottic compression were influenced by vocal effort and diagnosis of pMTD. Increased vocal effort was associated with lower CPP, particularly after vocal load, and decreased mediolateral supraglottic compression in the pMTD group.

Relationships Among Stimulability Testing, Patient Factors, and Voice Therapy Compliance.

OBJECTIVE: Voice stimulability testing to determine voice therapy efficacy and prognosis is commonly used during the voice evaluation, but little is known about how patient factors (eg, voice diagnosis, dysphonia severity) can influence stimulability outcomes. The predictability of voice therapy success with different stimulability facilitating techniques (eg, hums, pitch glides) is also unknown. The goals of this study were to identify relationships between patient factors, voice therapy compliance, and stimulability testing. METHODS: A retrospective chart review was conducted on 50 patients who were seen for their initial voice therapy evaluation at the UT Southwestern Clinical Center for Voice Care. Chart review included documentation of the stimulability tasks that yielded/did not yield voice changes, level of stimulability, voice diagnosis, clinician-rated auditory-perceptual analysis of vocal quality, therapy attendance, and compliance with voice therapy recommendations. Statistical analysis was conducted to determine whether the types of facilitating techniques, voice diagnosis, and dysphonia severity could predict how stimulable patients were and whether any stimulability techniques could predict voice therapy attendance and compliance. RESULTS: Patients diagnosed with functional voice disorders (eg, muscle tension dysphonia) were 11 times more likely to be stimulable for voice improvements than patients with neurological voice disorders (eg, vocal fold paralysis). Patients with lower dysphonia severity were more likely to be stimulable than patients with high dysphonia severity. Specific facilitating voice tasks did not predict the level of stimulability. Stimulability level was not predictive of therapy attendance or compliance with therapy recommendations. CONCLUSIONS: Voice diagnosis and severity of dysphonia influenced stimulability levels. However, voice stimulability was not predictive of voice therapy attendance or compliance, and no specific facilitative task predicted the level of stimulability. Future investigations should focus on other means of measuring a patient's motivation for change and on the predictive power of stimulability testing on voice therapy outcomes.

Relationships Between Laryngoscopic Analysis Metrics of Supraglottic Compression and Vocal Effort in Primary Muscle Tension Dysphonia.

PURPOSE: Supraglottic compression is thought to underlie vocal effort in patients with primary muscle tension dysphonia (pMTD). However, the relationship between supraglottic compression and vocal effort in this clinical population remains unclear. Gold standard laryngoscopic assessment metrics for supraglottic compression are also lacking. The goals of this study were to identify metrics proposed in the literature that could distinguish patients diagnosed with pMTD from typical voice users and determine their relationships to the vocal effort. METHODS: Flexible laryngeal endoscopy was performed on 50 participants (25 pMTD, 25 controls). The presence of supraglottic compression was characterized using a categorical (nominal) scale and severity was quantified on ordinal and continuous scales. The three laryngoscopic metrics were correlated with self-perceived ratings of vocal effort on a 100 mm visual analog scale. RESULTS: Inter-rater reliability was strongest for the continuous scale (P's < 0.0001) compared to categorical (P's < 0.001) and ordinal (P's < 0.001) scales. The presence of different supraglottic compression patterns varied in both groups, and there were no significant group differences on categorical (P's > 0.05) scales. Mediolateral (M-L) supraglottic compression was significantly greater in the pMTD group (P < 0.0001), and anteroposterior (A-P) compression was significantly greater in the control group (P = 0.001) using continuous scales. There were no significant relationships between any of the three laryngoscopic metric types and vocal effort ratings (P's > 0.05), except for a significantly positive relationship between anterior-posterior compression on the ordinal scale and vocal effort in the control group (P = 0.047). CONCLUSIONS: Continuous scales are reliable and valid for distinguishing individuals with pMTD from those without voice disorders, especially occupational voice users. M-L supraglottic compression may be a better indicator of pMTD than A-P compression. However, the poor correlation between supraglottic compression and vocal effort suggests that one may not influence the other. Future studies should focus on other mechanisms underlying vocal effort in patients with pMTD.

Patient Profiling: Determining the Effects of Patient Factors on Vocal Fatigue.

OBJECTIVES: The phenomenon of vocal fatigue and the types of patients that are at greatest risk for vocal fatigue are not fully understood. The goal was to investigate patient profiles such as voice disorder type, demographics (age and gender), singing identity, interoceptive awareness, and psychosocial impacts on the severity of vocal fatigue. STUDY DESIGN: Prospective cohort study. METHODS: Ninety-five subjects with voice disorders were asked to complete Part 1 of the Vocal Fatigue Index (VFI-Part1), the Voice Handicap Index-10 (VHI-10), and the Multidimensional Assessment of Interoceptive Awareness, version 2 (MAIA-2). The effects of voice disorder type (structural, neurological, functional), psychosocial impact, age, gender, self-reported singing identity, and interoceptive awareness on self-perceived vocal fatigue (VFI-Part1) were determined using multivariate linear regression. RESULTS: Vocal fatigue had a significant psychosocial impact on patients with voice disorders, as measured by the VHI-10 (P < 0.001). However, there were no significant effects of vocal fatigue across any of the three voice disorder types (P's >0.05). Age (P = 0.220), gender (P = 0.430), and self-identified singing experience (P = 0.360) also did not have significant effects on vocal fatigue. Additionally, there were no significant relationships between interoceptive awareness MAIA-2 sum scores (P = 0.056) or any of the MAIA-2 sub-scores (P's > 0.05) and vocal fatigue severity (VFI-Part1). CONCLUSION: Vocal fatigue has a significant psychosocial impact on patients with voice disorders. However, patient profiles, including voice disorder type, patient age, gender, singing identity, and level of interoceptive awareness do not appear to play a major role in vocal fatigue symptom reporting. These findings suggest caution should be exercised when attributing patient profiles to vocal fatigue presentation and severity. Studying pathophysiological mechanisms underlying vocal fatigue may help better distinguish unconscious bias in patient profiling from the etiology and severity of vocal fatigue.

Quantification of False Vocal Fold Hyperfunction During Quiet Breathing in Muscle Tension Dysphonia.

BACKGROUND/OBJECTIVES: False vocal fold (FVF) hyperfunction during phonation is thought to be a diagnostic sign of primary muscle tension dysphonia (pMTD). However, hyperfunctional patterns with phonation are also observed in typical speakers. This study tested the hypothesis that FVF posturing during quiet breathing, as measured by the curvature of FVF, could differentiate patients with pMTD from typical speakers. METHODS: Laryngoscopic images were collected prospectively in 30 subjects with pMTD and 33 typical speakers. Images were acquired at the end of expiration and maximal inspiration during quiet breathing, during sustained /i/, and during loud phonation before and after a 30-min vocal loading task. The FVF curvature (degree of concavity/convexity) was quantified using a novel curvature index (CI, >0 for hyperfunctional/convex, <0 for "relaxed"/concave) and compared between the two groups. RESULTS: At end-expiration, the pMTD group adopted a convex FVF contour, whereas the control group adopted a concave FVF contour (mean CI 0.123 [SEM 0.046] vs. -0.093 [SEM 0.030], p = 0.0002) before vocal loading. At maximal inspiration, the pMTD group had a neutral/straight FVF contour, whereas the control group had a concave FVF contour (mean CI 0.012 [SEM 0.038] vs. -0.155 [SEM 0.018], p = 0.0002). There were no statistically significant differences in FVF curvature between groups in either the sustained voiced or loud conditions. Vocal loading did not change any of these relationships. CONCLUSIONS: A hyperfunctional posture of the FVFs during quiet breathing especially at end-expiration may be more indicative of a hyperfunctional voice disorder than supraglottic constriction during voicing. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:3449-3454, 2023.

Extrinsic Laryngeal Muscle Tension in Primary Muscle Tension Dysphonia with Shear Wave Elastography.

OBJECTIVES: It has been assumed that patients with primary muscle tension dysphonia (pMTD) have more extrinsic laryngeal muscle (ELM) tension, but tools to study this phenomenon lack. Shear wave elastography (SWE) is a potential method to address these shortcomings. The objectives of this study were to apply SWE to the ELMs, compare SWE measures to standard clinical metrics, and determine group differences in pMTD and typical voice users before and after vocal load. METHODS: SWE measurements of the ELMs from ultrasound examinations of the anterior neck, supraglottic compression severities from laryngoscopic images, cepstral peak prominences (CPP) from voice recordings, and self-perceptual ratings of vocal effort and discomfort were obtained in voice users with (N = 30) and without (N = 35) pMTD, before and after a vocal load challenge. RESULTS: ELM tension significantly increased from rest-to-voiced conditions in both groups. However, the groups were similar in their ELM stiffness levels at SWE at baseline, during vocalization, and post-vocal load. Levels of vocal effort and discomfort and supraglottic compression were significantly higher and CPP was significantly lower in the pMTD group. Vocal load had a significant effect on vocal effort and discomfort but not on laryngeal or acoustic patterns. CONCLUSION: SWE can be used to quantify ELM tension with voicing. Although the pMTD group reported significantly higher levels of vocal effort and vocal tract discomfort and, on average, exhibited significantly more severe supraglottic compression and lower CPP values, there were no group differences in levels of ELM tension using SWE. LEVEL OF EVIDENCE: 2 Laryngoscope, 133:3482-3491, 2023.

Use of Motion Capture Technology to Study Extrinsic Laryngeal Muscle Tension and Hyperfunction.

OBJECTIVES: Patients with primary muscle tension dysphonia (pMTD) commonly report paralaryngeal pain and discomfort, and extrinsic laryngeal muscle (ELM) tension and hyperfunction are commonly implicated. However, quantitative physiological metrics to study ELM movement patterns for the characterization of pMTD diagnosis and monitoring of treatment progress are lacking. The objectives of this study were to validate motion capture (MoCap) technology to study ELM kinematics, determine whether MoCap could distinguish ELM tension and hyperfunction between individuals with and without pMTD, and investigate relationships between common clinical voice metrics and ELM kinematics. METHODS: Thirty subjects (15 with pMTD and 15 controls) were recruited for the study. Sixteen markers were placed on different anatomical landmarks on the chin and anterior neck. Movements across these regions were tracked during four voice and speech tasks using two three-dimensional cameras. Movement displacement and variability were determined based on 16 key-points and 53 edges. RESULTS: Intraclass correlation coefficients demonstrated high intra- and inter-rater reliability (p's < 0.001). Other than greater movement displacements around the thyrohyoid space during longer phrasing (reading passage, 30-s diadochokinetics) and more movement variability in patients with pMTD, kinematic patterns between groups were similar across the 53 edges for the four voice and speech tasks. There were also no significant correlations between ELM kinematics and standard voice metrics. CONCLUSION: Results demonstrate the feasibility and reliability of MoCap for the study of ELM kinematics. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:3472-3481, 2023.

Interoception, Voice Symptom Reporting, and Voice Disorders.

OBJECTIVES: Interoception may play a role in how individuals perceive their voice disorder. The first objective of this study was to investigate relationships between interoception and voice disorder class (functional, structural, neurological). The second objective was to determine relationships between interoception and voice-related outcome measures between patients with functional voice and upper airway disorders and typical voice users. The third objective was to determine whether patients with primary muscle tension dysphonia (a type of functional voice disorder) had different levels of interoceptive awareness than typical voice users. STUDY DESIGN: Prospective cohort study. METHODS: One hundred subjects with voice disorders completed the multidimensional assessment of interoceptive awareness-2 (MAIA-2). Voice diagnosis and singing experience were also acquired from each patient's medical chart. Voice handicap (VHI-10) and Part 1 of the vocal fatigue index (VFI-Part1) scores were obtained from patients diagnosed with functional voice and upper airway disorders. MAIA-2, VHI-10, VFI-Part1, and singing experience were also obtained from 25 typical voice users. Multivariable linear regression models were used to assess the association between response variables and voice disorder class, adjusting for singing experience, gender, and age. RESULTS: There were no significant group differences between voice disorder class (functional, structural, neurological) after adjusting for multiple comparisons. Participants with functional voice and upper airway disorders who scored significantly higher on the VHI-10 and VFI-Part1 had lower Attention Regulation sub-scores on the MAIA-2 (P's<0.05). Patients with primary muscle tension dysphonia scored significantly lower on the Emotional Awareness MAIA-2 subscale than typical voice users (P=0.005). CONCLUSION: Patients with functional voice disorders with lower capabilities to attend to body sensations may score higher on voice-related patient-reported outcome measures, like the VHI-10 and VFI-Part1. Patients with primary muscle tension dysphonia may also have lower capabilities in processing their body sensations than typical voice users.

Characterization of Primary Muscle Tension Dysphonia Using Acoustic and Aerodynamic Voice Metrics.

OBJECTIVES/HYPOTHESIS: The objectives of this study were to (1) identify optimal clusters of 15 standard acoustic and aerodynamic voice metrics recommended by the American Speech-Language-Hearing Association (ASHA) to improve characterization of patients with primary muscle tension dysphonia (pMTD) and (2) identify combinations of these 15 metrics that could differentiate pMTD from other types of voice disorders. STUDY DESIGN: Retrospective multiparametric METHODS: Random forest modeling, independent t-tests, logistic regression, and affinity propagation clustering were implemented on a retrospective dataset of 15 acoustic and aerodynamic metrics. RESULTS: Ten percent of patients seen at the New York University (NYU) Voice Center over two years met the study criteria for pMTD (92 out of 983 patients), with 65 patients with pMTD and 701 of non-pMTD patients with complete data across all 15 acoustic and aerodynamic voice metrics. PCA plots and affinity propagation clustering demonstrated substantial overlap between the two groups on these parameters. The highest ranked parameters by level of importance with random forest models-(1) mean airflow during voicing (L/sec), (2) mean SPL during voicing (dB), (3) mean peak air pressure (cmH2O), (4) highest F0 (Hz), and (5) CPP mean vowel (dB)-accounted for only 65% of variance. T-tests showed three of these parameters-(1) CPP mean vowel (dB), (2) highest F0 (Hz), and (3) mean peak air pressure (cmH2O)-were statistically significant; however, the log2-fold change for each parameter was minimal. CONCLUSION: Computational models and multivariate statistical testing on 15 acoustic and aerodynamic voice metrics were unable to adequately characterize pMTD and determine differences between the two groups (pMTD and non-pMTD). Further validation of these metrics is needed with voice elicitation tasks that target physiological challenges to the vocal system from baseline vocal acoustic and aerodynamic ouput. Future work should also place greater focus on validating metrics of physiological correlates (eg, neuromuscular processes, laryngeal-respiratory kinematics) across the vocal subsystems over traditional vocal output measures (eg, acoustics, aerodynamics) for patients with pMTD. LEVEL OF EVIDENCE: II.

Acoustic Measures of Voice and Physiologic Measures of Autonomic Arousal During Speech as a Function of Cognitive Load in Older Adults.

OBJECTIVES/HYPOTHESIS: The purpose of this study was to determine the relationships among cognitive loading, autonomic arousal, and acoustic measures of voice in healthy older adults. STUDY DESIGN: Prospective and observational. METHODS: Twelve healthy older adults (six females) produced a sentence containing an embedded Stroop task in each of two cognitive load conditions: congruent and incongruent. Three physiologic measures of autonomic arousal (pulse volume amplitude, pulse period, and skin conductance response amplitude) and four acoustic measures of voice (cepstral peak prominence, low-to-high spectral energy ratio, fundamental frequency, and sound pressure level) were analyzed in each cognitive load condition. RESULTS: A logistic regression model was used to predict the cognitive load condition using participant as a categorical predictor and the four acoustic measures and three autonomic measures as continuous predictors. Skin conductance response amplitude and pulse volume amplitude were both predictive of cognitive load; however, no acoustic measures of voice were statistically significant predictors of cognitive load for older adults. CONCLUSIONS: These findings support the idea that increased cognitive load is associated with increased autonomic nervous system activity in older adults. The lack of changes in acoustic measures of voice with increased cognitive load may result from age-related changes in vocal quality and speech subsystems.

Clinical Characteristics and Effects of Vocal Demands in Occupational Voice Users With and Without Primary Muscle Tension Dysphonia.

OBJECTIVES: The objectives of this study were to (1) compare laryngeal configuration patterns in occupational voice users with and without primary muscle tension dysphonia (pMTD), (2) characterize laryngeal configurations in relation to other clinical metrics (acoustic, perceptual), and (3) determine the effects of vocal demands (load) on these clinical parameters. METHODS: Thirty subjects (15 pMTD, 15 control) were recruited for the study. Laryngoscopic examinations and voice samples for sustained /i/ were obtained before and after a half hour vocal load task. Subjects rated their vocal effort and discomfort before and after the vocal load. Laryngeal configurations were analyzed subjectively with dichotomous and categorical rating scales and hyper function severity quantified (endolaryngeal area outlets). Overall dysphonia severity and vocal instability of each voice sample was rated on 100mm visual analog scales and cepstral peak prominence (CPP) extracted from each voice sample. RESULTS: Laryngeal configurations between groups or vocal load condition were not distinguishable with any of the dichotomous, categorical, or quantitative laryngeal metrics. Vocal effort and discomfort ratings were significantly higher in the pMTD group compared to the control group. Vocal load also had significant effects across groups on vocal effort and vocal tract discomfort ratings. Although CPP values fell within the normal range in both groups, CPP was significantly lower in the pMTD group at both pre-load and post-load time points. Auditory-perceptual ratings were also significantly worse in the pMTD group. Vocal load did not have a significant effect on acoustic or auditory-perceptual measures. CONCLUSION: Similar laryngeal configurations between groups at both pre- and post-vocal load suggests classic patterns of laryngeal "hyperadduction" may occur variably in occupational voice users and may not be indicative of pMTD pathophysiology. Greater vocal effort, discomfort, instability, and perturbation within the vocal system may better define pMTD than laryngeal configuration in occupational voice users with pMTD.

Potential Biophysiological Mechanisms Underlying Vocal Demands and Vocal Fatigue.

Patients with complaint of vocal fatigue have perceptual, acoustic, and aerodynamic outcomes that are heterogeneous in nature. One reason may be due to different underlying biophysiological mechanisms that lead to these heterogeneous clinical presentations. Five potential mechanisms are proposed: neuromuscular, metabolic, vocal tissue, afferent, and central neural. Analytical frameworks and study designs to study these mechanisms are also addressed. A better understanding of biophysiological mechanisms of vocal fatigue can improve precision of therapeutic approaches. It can also help shift management from symptom-based to etiology-focused approaches for vocal fatigue.

Laying the Groundwork to Study the Heterogeneous Nature of Vocal Fatigue.

Vocal fatigue has remained an elusive construct-despite its significant impact on communication, vocation, and quality of life. Current frameworks define vocal fatigue in the context of vocal demands and vocal demand-responses. However, the impact of factors like individuals' baseline vocal fitness and perception of the demand are not well understood. What is also not well understood are the effects of specific vocal demand ingredients on an individual's vocal demand responses. Furthermore, current outcome measures utilized to capture vocal fatigue lack sensitivity and underlying mechanisms are poorly understood. These gaps have led to inconclusive next steps in how to best define, assess, monitor, and manage vocal fatigue. A conceptual framework is needed to study and better understand vocal fatigue constructs. Such a framework should consider the individual's baseline physiology, psychology, key vocal demand ingredients, and biophysiological mechanisms underlying demand responses. The objective of this paper is to help the reader better understand the complex and heterogeneous nature of vocal fatigue and its impact on reliable assessment and monitoring. Future studies will require better elucidation of vocal demand ingredients, will need more sensitive vocal demand response measures, and will need to take in to account an individual's baseline physiology and psychological factors.

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.

Proteomic Characterization of Senescent Laryngeal Adductor and Plantaris Hindlimb Muscles.

OBJECTIVES: The goals of this study were to 1) compare global protein expression in muscles of the larynx and hindlimb and 2) investigate differences in protein expression between aged and nonaged muscle using label-free global proteomic profiling methods. METHODS: Liquid chromatography-mass spectrometry (LC-MS/MS) analysis was performed on thyroarytenoid intrinsic laryngeal muscle and plantaris hindlimb muscle from 10 F344xBN F1 male rats (5 old and 5 young). Protein expression was compared and pathway enrichment analysis performed for each muscle type (larynx and limb) and age group (old and young muscle). RESULTS: Over 1,000 proteins were identified in common across both muscle types and age groups using LC-MS/MS analysis. Significant age-related differences were seen across 107 proteins in plantaris hindlimb and in 19 proteins in thyroarytenoid laryngeal muscle. Bioinformatic and enrichment analysis demonstrated protein differences between the hindlimb and larynx may relate to immune and stress redox responses and RNA repair. CONCLUSION: There are clear differences in protein expressions between the laryngeal and hindlimb skeletal muscles. Initial analysis suggests differences between the two muscle groups may relate to stress responses and repair mechanisms. Age-related changes in the thyroarytenoid appear to be less obvious than in the plantaris. Further in-depth study is needed to elucidate how aging affects protein expression in the laryngeal muscles. LEVEL OF EVIDENCE: NA Laryngoscope, 132:148-155, 2022.

Metabolomic Expression of Laryngeal and Hindlimb Muscles in Adult versus Senescent Rats.

OBJECTIVES: (1) Determine the feasibility of obtaining a global, unbiased metabolomic profile on laryngeal muscle in a rat model; (2) evaluate the impact of biological aging on the laryngeal metabolome; and (3) characterize biochemical expression differences between aged and non-aged laryngeal and hindlimb muscle. METHODS: Thyroarytenoid laryngeal muscle and plantaris hindlimb muscle were harvested from 5 young adult (9 months old) and 5 older adult (32 months old) F344BN rats. Tissue was processed and analyzed using LC-MS methods. Detected metabolites were compared to widely used metabolite databases and KEGG pathway enrichment was performed on significant metabolites. RESULTS: The greatest differences in metabolite expression were between laryngeal and limb muscle with 126 different metabolites found between laryngeal and limb within the young group and 149 different metabolites within the old group. Significant hits between muscle groups highlighted amino acid differences between these tissues. There were more robust differences with age in limb muscle compared to laryngeal muscle. CONCLUSIONS: Amino acid metabolism is a key difference between muscles of the limbs and larynx. Due to the number of differentially expressed metabolites between the 2 muscle groups, caution should be exercised when applying skeletal limb muscle physiology and biology concepts to the vocal muscles in both aged and non-aged musculoskeletal systems. Mechanisms underlying less robust effects of age on laryngeal muscle compared to limb muscle require elucidation.

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.

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.