Modeling Laryngeal Dystonia through Spectral Analyses of Vocalizations in a Dystonia Mouse Model

Abstract

Laryngeal dystonia is a task-specific, focal dystonia that disrupts vocal-motor control and significantly alters quality of life through impaired communication. Despite its early onset in many hereditary dystonias, effective treatments remain limited, in part due to the lack of a preclinical model that captures its circuit-level pathophysiology. Our experiment evaluates ultrasonic vocalizations (USVs) in Ptf1a ^Cre/+;Vglut2 ^fl/fl mice, a cerebellum-specific generalized dystonia model, to assess translational relevance for laryngeal dystonia. At postnatal day 9, mutant mice demonstrated statistically significant reductions in total USV count, relative count of complex calls, and key spectral parameters-especially frequency modulation and power-mirroring phonatory abnormalities seen in human patients. Cluster analyses further revealed impaired vocal burst initiation, suggesting disrupted cerebellar coordination of temporal vocal-motor output. These findings support the model's construct and face validity for cerebellar contributions to disordered phonation. By revealing these potential translational biomarkers, our study establishes a foundational platform for future mechanistic and interventional research in laryngeal dystonia.

Keywords: cerebellar dysfunction; laryngeal dystonia; mouse model; spasmodic dysphonia; translational neuroscience; ultrasonic vocalizations.

Figure 1.. Example spectrograms of categorized USV call types recorded from P9 mouse pups.

Each panel shows an example USV spectrogram annotated by the Metris Sonotrack software according to spectral features which include frequency (Y-axis, kHz), time (X-axis, ms), and power intensity (color scale, dB). Calls are categorized based on frequency modulation, shape, and complexity. These spectrograms are intended as qualitative examples only. All USV call types were categorized according to Metris Sonotrack Call Classification which is based upon call classification principles described in Portfors (2007). Additionally, ‘syllables’ refer to individual vocal elements within a call and are synonymous with Sonotrack’s definition of ‘elements’ (Portfors 2007): (A) Short Calls are brief, simple calls with short duration <15 ms. These calls are often isolated with little to no modulation. One Syllable Calls include: (B1) Flat Calls are calls with a nearly constant frequency throughout. This indicates no major pitch change, producing a monotone tone. (B2) Up Calls where the frequency increases over time corresponding to a rising pitch. (B3) Down Calls where the frequency decreases over time corresponding to a falling pitch. (B4) Chevron Calls are V- or inverted V-shaped calls with a sharp rise and fall (or fall and rise) in frequency. They are often brief and energetic. (B5) U-Shape Calls are a descending then ascending smooth curve in frequency. Two Syllable Calls include: (C1) Trailing Calls end with a long, fading frequency tail. (C2) Step Down Calls have one or more abrupt drops in frequency and resemble a stair-step downward. (C3) Step Up Calls abruptly shift upwards in frequency like stair-steps going upward. 3–4 Syllable Calls include: (D1) Step Double Calls are composed of two sequential step-like changes in frequency, creating a double-stepped shape. (D2) Complex-3 Calls contain three distinct modulations or frequency shifts and suggest more sophisticated communication. (D3) Complex-4 Calls are four-part calls with varying pitch directions or shapes, reflecting more nuanced or emotionally complex vocalizations. 5+ Syllable Calls include: (E1) Complex-5 Calls are five-segment calls with multiple frequency modulations. They are typically rare. (E2) Complex-5+ Calls are calls with more than five modulated components which make them highly elaborate.

Figure 2:. Quantitative analysis of USV call types reveals fewer total vocalizations and a statistically significant reduced percentage of the most complex calls in dystonic pups.

Graphs represent individual data points and show group means for total USV count and call type distributions grouped by syllable number. Individual data points represent biological replicates (one pup; n=27 Vglut2^fl/fl controls, 23 Ptf1a^Cre/+;Vglut2^fl/fl mutants; experiment performed once per pup). (A) Total number of USVs emitted per pup. (B1, C1, D1, E1, F1) Number of Short, One-, Two-, Three to Four-, and Five+ syllable calls emitted per pup, respectively. (B2, C2, D2, E2, F2) Percentage of Short, One-, Two-, Three to Four-, and Five+ syllable calls emitted per pup, respectively. Syllable categorization of call types is explained in Figure 1. Statistical comparisons were performed using unpaired, two-tailed Mann-Whitney U tests. Statistically significant reductions were observed in total number of USVs, absolute numbers of all call type categories, and relative Five+ syllable calls in mutants compared to controls. Data suggests a marked reduction in both number and complexity of vocalizations in mutant animals. *p<0.05; **p<0.01; ***p<0.001.

Figure 3:. Analysis of acoustic properties of short calls indicates altered signal structure in dystonic pups.

Graphs represent individual data points and show group means for various acoustic parameters of short calls (<15 ms in duration). Individual data points represent biological replicates (one pup; n=27 Vglut2^fl/fl controls, 23 Ptf1a^Cre/+;Vglut2^fl/fl mutants; experiment performed once per pup). (A) Mean short call duration (ms). (B) Mean frequency average (kHz). (C1, C2) Relevant power parameters of mean power average (dB) and mean power maximum (dB), respectively. (D1-D3) Frequency modulation parameters of mean maximum downward change in frequency (kHz), mean maximum upward change in frequency (kHz), and mean rate of frequency change (kHz s⁻¹), respectively. Statistical comparisons were performed using unpaired, two-tailed Mann-Whitney U tests. Statistically significant differences were detected in upward frequency change and maximum power, indicating acoustic alterations in short calls emitted by mutant pups. *p<0.05; **p<0.01; ***p<0.001.

Figure 4:. Analysis of acoustic properties of long calls shows altered signal structure in dystonic pups.

Graphs represent individual data points and show group means for various acoustic parameters of long calls (>15 ms in duration). Individual data points represent biological replicates (one pup; n=27 Vglut2^fl/fl controls, 23 Ptf1a^Cre/+;Vglut2^fl/fl mutants; experiment performed once per pup). (A) Mean long call duration (ms). (B) Mean frequency average (kHz). (C1, C2) Relevant power parameters of mean power average (dB) and mean power maximum (dB), respectively. (D1-D3) Frequency modulation parameters of mean maximum downward change in frequency (kHz), mean maximum upward change in frequency (kHz), and mean rate of frequency change (kHz s⁻¹), respectively. Statistical comparisons were performed using unpaired, two-tailed Mann-Whitney U tests. A statistically significant difference was detected in mean rate of frequency change, indicating acoustic alterations in long calls emitted by mutant pups. *p<0.05; **p<0.01; ***p<0.001.

Figure 5:. Cluster analysis of USV patterns reveals altered vocal grouping behavior in dystonic pups.

Graphs represent individual data points and show group means for cluster-based metrics derived from USVs. Individual data points represent biological replicates (one pup; n=27 Vglut2^fl/fl controls, 23 Ptf1a^Cre/+;Vglut2^fl/fl mutants; experiment performed once per pup). A cluster was defined as a group of 2 or more unique calls where the start time of each call occurred within 0.5 s of the end time of the previous call (this also defines our inter-call interval). (A) Number of detected USV clusters per pup. (B) Mean number of calls per cluster. (C) Mean cluster inter-call interval (s) within clusters. (D) Mean cluster duration (s). Statistical comparisons were performed using unpaired, two-tailed Mann-Whitney U tests. A statistically significant reduction in the number of USV clusters in mutant pups compared to controls was observed, suggesting altered temporal organization of vocalizations. *p<0.05; **p<0.01; ***p<0.001.