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The work included in this paper was supported in part by the National Institute on Deafness and Other Communication Disorders (NIDCD) of the National Institutes of Health (NIH) under award number R01DC0020179, an American Speech-Language Hearing Foundation New Investigators Research Grant (A.J.S.), Collins Medical Trust Grants (A.J.S.), and startup funds provided to A.J.S. by Purdue University.

<ol>
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There are no relevant conflicts of interest.

The present protocol provides a reproducible approach for eliciting appetitive 50 kHz ultrasonic vocalizations (USVs) using a food-reward paradigm, with several critical steps required to ensure consistent and interpretable outcomes. Foremost, animal handling and acclimation are essential for reducing stress-related variability in vocal output13. Daily handling under controlled conditions and systematic habituation to the testing chamber promote reliable anticipatory behavior and stable USV production across sessions. Behavioral conditioning to the food reward is another key step; inconsistent or insufficient conditioning can result in low vocalization rates or high inter-animal variability6,34. Maintaining consistent personnel and testing conditions, including recording during the dark cycle or under red-light conditions, eliminating environmental noise sources, and standardizing chamber setup, is equally critical for optimizing signal quality and minimizing artifacts5. Without these controls, it becomes difficult to determine whether changes in USVs reflect behavior or simply noise introduced by the recording environment.
This protocol is designed with flexibility to accommodate modifications based on specific experimental needs. For example, the type and novelty of palatable food rewards can be adjusted to maintain motivation across repeated sessions, particularly in longitudinal or disease-model studies. The duration of acclimation and recording sessions can also be modified depending on species, strain, or experimental constraints. Troubleshooting common issues primarily involves addressing low USV output or excessive noise. Low vocalization rates can often be improved by extending conditioning periods, increasing reward salience, or confirming that animals are tested during their active phase. Acoustic artifacts can be minimized by ensuring proper chamber cleaning between animals, eliminating bedding and extraneous materials, and verifying consistent microphone positioning and recording parameters. Additionally, randomizing animals across recording chambers helps mitigate potential chamber-specific variability and enhances experimental rigor.
A major strength of this method lies in its alignment with recent efforts to standardize laboratory rat and mouse USV methodologies, as highlighted by Shembel et al. (2025)5. Standardization across elicitation paradigms, recording environments, and analysis pipelines is essential to improve reproducibility and enable cross-study comparisons. This protocol directly addresses these priorities by implementing structured acclimation procedures, controlled stimulus presentation, and consistent recording conditions. The use of a food-reward paradigm supports reliable and repeatable elicitation of USVs and reduces behavioral variability associated with social or mating-based paradigms5,28. Recording during the animals&#8217; active phase and minimizing environmental noise further aligns with best-practice recommendations to optimize signal quality and biological relevance5. In addition, the use of defined acoustic outcome measures, including USV number, rate, frequency range, duration, maximum power, and complexity ratio, supports transparent and standardized analysis practices, which are critical given the variability in classification approaches across the field5.
The interpretation of USVs also differs meaningfully between rats and mice and should be considered when applying this protocol across species. In rats, 50 kHz ultrasonic vocalizations are well-established markers of positive affect and reward-related states. In contrast, mouse USVs are more heterogeneous in both acoustic structure and behavioral context, and USVs within similar high-frequency ranges do not map as directly onto affective valence. Instead, mouse USVs may reflect a combination of social communication, arousal, and context-dependent behavioral states. Accordingly, the present protocol treats USVs as species-specific behavioral readouts rather than a singular affective marker across laboratory rats and mice. Despite the strengths, several limitations should be considered when applying this method. While the food-reward paradigm reduces hormonal and social confounds, it specifically captures appetitive, reward-driven vocalizations and may not generalize to other behavioral contexts such as distress or social communication. Motivation for food rewards may also vary across animals, experimental conditions, or disease states, potentially influencing vocal output independently of vocal motor function6,34. Importantly, the representative datasets presented here are derived from a single strain of laboratory rats and a single strain of laboratory mice, which limits generalizability. Although the protocol is designed to be applicable across laboratory rats and mice, species- and strain-specific differences in vocal behavior, conditioning response, and acoustic characteristics should be expected, and broader validation across strains and experimental contexts is warranted. Additionally, although automated classification software facilitates high-throughput analysis, variability in detection thresholds and classification algorithms may affect comparability across studies. The protocol is presented using an integrated system for reproducibility, but is intended to be broadly adaptable across ultrasonic recording platforms with equivalent specifications. Careful validation of analysis parameters and consistent reporting of acoustic features are therefore necessary to ensure data reliability. Finally, the requirement for specialized ultrasonic recording equipment and sound-attenuating chambers may limit accessibility for some laboratories.
Importantly, the representative results included in this protocol incorporate a combination of heterogenous datasets, including single-session exemplars, pilot training data, and illustrative experimental outcomes. These examples are intended to demonstrate the range of expected outputs and common analytical approaches rather than to provide definitive biological conclusions. As such, these datasets should be interpreted as methodological demonstrations rather than hypothesis-driven results. Additionally, references to in-press or unpublished datasets are included to illustrate potential applications of the protocol, but should not be interpreted as validated outcomes within the context of this methods-focused report.
The significance of this method lies in its ability to provide a controlled, non-social paradigm for studying vocal behavior while adhering to emerging standards for methodological rigor and reproducibility. By eliminating mating-related variables and standardizing elicitation and recording conditions, this approach offers clear advantages over traditional paradigms, particularly when the goal is to isolate physiological influences on vocal production independent of social context. The protocol is particularly well-suited for applications in neurodegenerative disease models, endocrine manipulation studies, and investigations of cranial sensorimotor function, where minimizing confounding variables is critical. More broadly, this method contributes to ongoing efforts to harmonize USV research practices and provides a standardized framework for eliciting and quantifying vocal behavior. While such standardization is a necessary step toward evaluating the potential of USVs as translational biomarkers of affective and sensorimotor function, the present protocol does not, in itself, establish biomarker validity. Rather, it enables the consistent data acquisition and analysis needed for future studies to rigorously test translational relevance.

Ultrasonic vocalizations (USVs) in laboratory rats and mice are widely used as experimental measures of vocal behavior and affective state and provide a valuable model for studying vocal communication under controlled conditions. Rats and mice produce ultrasonic range vocal signals across a variety of biologically relevant contexts, including mating, distress signaling, play, and social communication1,2,3,4,5. As USVs reflect both physiological and behavioral states, they provide a sensitive and quantifiable outcome measure in studies of emotion, social behavior, and brain&#8211;behavior relationships5. Laboratory rats and mice produce communicative USVs at high frequencies ranging 30&#8211;90 kHz in rats and 40&#8211;120 kHz in mice, often referred to as &#8220;50 kHz USVs.&#8221; In rats, 50 kHz USVs are commonly associated with positive affective states, reward anticipation, and social approach behaviors2,5,6,7. Mice produce 50 kHz USVs in comparable social and affective contexts, but exhibit greater variability in structure and are strongly dependent on behavioral context and social cues8,9,10,11,12. As such, USVs have been widely used in psychosocial and psychobiological research to study emotional processing, social interactions, learning paradigms, and the effects of pharmacological manipulations on the nervous system5,11,13,14,15,16,17. More recently, USVs have also been adopted as outcome measures in studies investigating neurological disease, neurodegeneration, developmental disorders, and cranial sensorimotor dysfunction relevant to human communication disorders5,18,19,20,21,22.
USV production occurs within the larynx and requires precise neuromuscular coordination of the intrinsic laryngeal muscles housed within a cartilaginous framework23,24,25. Motor patterns controlling muscles such as the thyroarytenoid and cricothyroid regulate vocal fold position and glottal configuration during sound production26. Unlike human phonation, which relies on flow-induced vocal fold oscillation, USVs are produced through an aerodynamic whistle mechanism in which high-velocity airflow exits the glottis and interacts with laryngeal structures to generate ultrasonic sound23,24,25,27. Consequently, the acoustic features of USVs reflect coordinated neuromuscular control of the larynx, vocal tract configuration, and subglottal pressure supply rather than vibratory biomechanics of the vocal fold mucosa. For experimental measurement, USVs are primarily defined and analyzed based on acoustic features captured during recordings. These USV acoustic features, including frequency, duration, and intensity, can be extracted using automated or semi-automated analysis pipeline, which forms the basis for quantitative comparisons across experimental conditions5.
A substantial body of literature has used USVs to examine hormonal influences on vocal behavior across laboratory rats and mice. Many studies have demonstrated that reproductive hormones (e.g., estrogen and progesterone) modulate USV production and acoustic characteristics, particularly within mating contexts4,28,29. For example, estrous cycle stage, ovariectomy, and hormone replacement have been shown to alter USV rates and acoustic features in both female rats and mice, including modulation by estrous state and reproductive hormonal cues in mice28,30,31,32,33. However, these investigations have largely relied on mating-based paradigms, such as the presentation of an estrous female to a male rat or mouse, which robustly elicits vocalizations but introduces substantial variability related to sexual motivation, social experience, and endocrine state. While these paradigms are highly effective for eliciting large numbers of USVs, the behavioral and hormonal complexity inherent in reproductive contexts can complicate interpretation in studies focused on quantifiable vocal output or controlled experimental manipulation. As such, differences in social interaction, sexual receptivity, and motivational state may independently influence USV production, making it difficult to isolate the physiological variables under investigation.
Outside of reproductive USV elicitation contexts, fewer standardized approaches exist for examining USV production under controlled, non-social conditions in both rats and mice. Emerging work suggests that hormonal status can influence USV production even in non-reproductive contexts. For instance, estradiol replacement has been reported to alter USV acoustic features and rates depending on the social context in which vocalizations are recorded28,30. In one study examining USVs outside mating paradigms, the estrous cycle stage influenced acoustic parameters in both social isolation and dyadic interaction conditions28. However, variability across recording context, environmental conditions, and elicitation paradigms remains a major challenge for reproducibility and cross-study comparison.
Given these limitations, experimental paradigms that minimize social and reproductive confounds are important for studying USVs in a controlled and reproducible manner. Food-associated reward paradigms provide a useful alternative approach for eliciting appetitive 50 kHz USVs. Rats, and to a lesser extent mice, produce anticipatory USVs in response to reward-associated cues, including access to palatable food, reflecting motivational states and reward expectancy6,9,34. In this approach, animals are acclimated to the recording environment and conditioned to associate chamber exposure with a palatable food reward, resulting in consistent vocal output across sessions. Compared with mating-based paradigms, reward-driven elicitation enables precise standardization of environmental conditions, reduces variability related to social interaction or reproductive status, and can be applied across sexes and experimental models. Additionally, this approach is compatible with a range of ultrasonic recording systems and supports repeated measures designs. These features make this paradigm particularly well-suited for studies requiring consistent USV elicitation and quantitative comparison across conditions. This approach becomes especially important when trying to isolate relatively subtle changes in vocal output that would otherwise be masked by variability in social or reproductive behavior.
The overall goal of the present method is to provide a detailed and reproducible protocol for eliciting and quantifying appetitive ultrasonic vocalizations using a conditioned anticipatory food-reward paradigm. The protocol outlines procedures for animal handling, acclimation, and behavioral conditioning, followed by standardized USV recordings conducted in sound-attenuated chambers under controlled environmental conditions. A structured workflow for automated detection and classification is also described, enabling the extraction of USV-level acoustic parameters from each recording, including total USV number, rate, frequency range, duration, maximum power, and complexity. This methods-focused framework emphasizes reproducibility, standardization, and adaptability across laboratory settings and species, providing a practical pipeline for consistent USV data acquisition and analysis.
This protocol provides a standardized and reproducible approach for eliciting and recording appetitive USVs using an anticipatory food-reward paradigm, enabling detailed analysis of vocal behavior across experimental conditions. Although this protocol is applicable to both laboratory rats and mice, important species-specific differences should be considered. In rats, 50 kHz USVs are well-characterized and are commonly associated with positive affective states and reward anticipation. In mice, USVs are more variable in both structure and behavioral interpretation, are not reliably indicative of positive affective states, and should not be interpreted as direct proxies of emotional valence8,9. Additionally, acoustic features such as USV duration, frequency range, and modulation patterns differ between species, which may require adjustment of detection and classification parameters. Behavioral responses to conditioning may also vary, with rats generally exhibiting more robust and consistent appetitive vocalization in reward-based paradigms. Several steps in this protocol are critical for reliable USV elicitation (e.g., acclimation duration, reward pairing, and recording consistency), whereas other parameters (e.g., reward type, chamber type, and acquisition parameters) may be adjusted based on species, strain, or experimental design, provided that validation and parameter optimization are completed when adapting this protocol across laboratory rats and mice.

<table><tbody><tr><td>70% ethanol</td><td>Fisher Scientific</td><td>BP28184</td><td>Used to clean chamber between animals</td></tr><tr><td>Additional palatable treats (e.g., shelled peanuts)</td><td>Bio-Serv</td><td>F6711</td><td>Optional reward variation to sustain engagement</td></tr><tr><td>Animal transfer station (if used)</td><td>NUAIRE</td><td>AllerGard NU-621</td><td>Provides controlled handling environment during experimenter socialization (if applicable to your protocol)</td></tr><tr><td>Blackout curtain/sheet</td><td>Mainstays (Walmart)</td><td>https://www.walmart.com/ip/Mainstays-Super-Soft-Brushed-Microfiber-Bed-Sheet-Set-Rich-Black-King-4-Piece/12946065251</td><td>Used to eliminate ambient light and external noise disturbances during transfer</td></tr><tr><td>Cart (transport)</td><td>Metro</td><td>13-460-126</td><td>Supports cages during transfer</td></tr><tr><td>Computer/acquisition interface</td><td>Windows</td><td>10 Professional 64bit</td><td>Must meet Sonotrack software requirements; used for recording and data storage</td></tr><tr><td>Data storage device (external hard drive/server)</td><td>Glyph</td><td>BBPR4000</td><td>For secure storage of large acoustic files</td></tr><tr><td>Fruit Loops</td><td>Kellogg&#039;s</td><td>4119210032</td><td>Palatable food reward to maintain novelty and motivation</td></tr><tr><td>Garden Veggie Straws</td><td>Sensible Portions</td><td>829515300494</td><td>Palatable food reward to maintain novelty and motivation</td></tr><tr><td>Laboratory detergent (mild soap)</td><td>Dawn</td><td>91064</td><td>Used for post-session cleaning of cages and inserts</td></tr><tr><td>Paper towel</td><td>ToolBox</td><td>5520208</td><td>Used with ethanol for cleaning</td></tr><tr><td>Polycarbonate recording cages (no bedding)</td><td>UNO Life Science Solutions</td><td>31200260</td><td>Used withing recording chamber to minimize noise artifacts</td></tr><tr><td>PPE: disposable lab gown</td><td>Fisherbrand</td><td>12-893-005XXL</td><td>Prevents contamination and odor transfer</td></tr><tr><td>PPE: nitrile gloves</td><td>Ansell</td><td>19-168-967D</td><td>Prevents contamination and odor transfer</td></tr><tr><td>Red lighting system</td><td>Kenall</td><td>https://kenall.com/Animal-Research-Lab-Lighting#:~:text=Red%20LED%20lighting%20is%20ideal%20for%20animal%20research,for%20nocturnal%20animals%20such%20as%20mice%20and%20rats.</td><td>Low-visibility lighting to maintain reverse light cycle while allowing experimenter visibility</td></tr><tr><td>Reese&#039;s Puffs</td><td>General Mills</td><td>10016000275666</td><td>Palatable food reward to maintain novelty and motivation</td></tr><tr><td>SmartChamber for Mice and Rats</td><td>Metris</td><td>SC-B100</td><td>Sound-attenuating chamber for ultrasonic vocalization recording</td></tr><tr><td>Sonotrack Automated Call Classification software (v1.4)</td><td>Metris</td><td>ST-S400</td><td>Used for USV detection, classification, and export of acoustic parameters</td></tr><tr><td>Sonotrack Base configuration</td><td>Metris</td><td>ST-R2P0-M</td><td>Data acquisition hardware for ultrasonic recording</td></tr><tr><td>Sonotrack High-end Ultrasound Microphone</td><td>Metris</td><td>ST-B200</td><td>Frequency range ~15-125 kHz for rodent USV detection</td></tr><tr><td>Standard home cages</td><td>Innovive</td><td>M-BTM (Mouse); R-BTM-H (Rat)</td><td>Standard housing/transfer cages</td></tr><tr><td>Timer</td><td>Fisherbrand</td><td>06-664-251</td><td>Ensures standardized 10-min recording sessions</td></tr></tbody></table>

anticipatory food-reward elicitation of appetitive ultrasonic vocalizations in laboratory rats and mice

Anticipatory Food-Reward Elicitation of Appetitive Ultrasonic Vocalizations in Laboratory Rats and Mice

Ultrasonic vocalizations (USVs) in laboratory rats and mice provide a valuable model for investigating vocal function, affective behavior, and the underlying neural mechanisms that drive vocalization. Many commonly used USV elicitation paradigms rely on mating or social interaction, which can introduce hormonal and behavioral confounds that complicate interpretation in studies of aging, neurodegeneration, and endocrine modulation. To address these limitations, this article describes a food-reward USV elicitation paradigm that reliably evokes appetitive 50 kHz vocalizations in anticipation of palatable food. It includes standardized procedures for animal handling, acclimation, and behavioral conditioning to establish consistent anticipatory responses to a palatable reward. Animals undergo repeated exposure to the recording environment followed by reward delivery to promote stable vocal output across sessions. USVs are recorded in sound-attenuated chambers under controlled environmental conditions to ensure a high signal-to-noise ratio and reproducibility. A structured workflow for automated detection and classification is provided, which enables the extraction of key acoustic parameters, including the total number of USVs, rate, frequency range, duration, maximum power, and complexity. The protocol includes quality control steps to verify detection accuracy and minimize artifacts. By eliminating mating-related variables and standardizing recording conditions, this method provides a controlled approach for eliciting and quantifying appetitive 50 kHz vocalizations across repeated sessions. The paradigm is well-suited for investigating affective-motivational processes and cranial sensorimotor function and can be adapted for diverse experimental contexts, including studies of neurological disease, neurodegeneration, and hormone manipulation, with appropriate study-specific validation.

Successful implementation of this USV recording and analysis protocol yields high-quality recordings with minimal background noise, optimized experimental conditions, enabling reliable visualization and extraction of individual USVs. Under these conditions, animals produce measurable USVs during the 10 min recording period that are clearly visible on spectrograms and can be detected and classified using Call Classification Software. Representative recordings demonstrate distinct USV contours with well-defined acoustic contours, including flat and frequency-modulated USV structures, enabling accurate extraction of acoustic parameters (Figure 1).
Quantitative outputs from successful experiments include total USV number, USV rate (USVs/min), frequency range, duration, maximum power, and USV complexity ratio (simple vs. complex). In well-conditioned animals, total USV counts are consistent across recording days, and USV rates remain stable within individuals. Frequency range and duration parameters fall within expected species-specific ranges (e.g., 30–90 kHz band for rat USVs and 40–120 kHz band for mouse USVs) and paradigm with clear separation between simple and complex vocalizations. The complexity ratio provides an additional measure of vocal behavior, with shifts toward either simple or complex USVs that may reflect effects of experimental manipulations. Figure 2 showcases representative outputs from a single recording session to illustrate the classification and distribution of USVs across simple and complex USV types, frequency modulation patterns, and duration bins, and to demonstrate the successful detection and analysis of vocal behavior using this elicitation method. Specifically, Figure 2A displays the total number of identified USVs categorized into various simple and complex types; Figure 2B shows the distribution of USVs across frequency modulation patterns (flat, upward, downward, and complex profiles); and Figure 2C presents the distribution of USVs grouped by total duration.
Representative datasets from successful experiments demonstrate variability between animals at baseline and across experimental groups or time points. Data shown in Figure 3 are derived from an independent published dataset using 5-7-month-old Long-Evan rats and demonstrate how physiological state (e.g., hormonal or hydration manipulations) can be reflected in quantifiable acoustic USV features, such as duration, power, or frequency range37. These results are presented as examples of how the protocol enables the extraction and analysis of exported USV datasets from the software rather than as definitive evidence of biological effects. In contrast, Figure 4 presents unpublished pilot data from a single mouse strain (A53T alpha-synuclein overexpression mice) and age range (3–5 months old) undergoing a vocal training paradigm, in which no significant differences were observed between control and trained groups. This example demonstrates the ability of the protocol to capture both variable and null outcomes, which underscores the importance of study-specific optimization and validation. Together, these datasets illustrate the analytical outputs generated by the protocol across laboratory rats and mice.
In contrast, suboptimal recordings are characterized by excessive background noise, reduced spectrogram clarity, or inconsistent USV detection. For this protocol, recordings are considered suboptimal when visual inspection of the spectrogram indicates background noise that substantially obscures USV structure within the 20–100 kHz range, or when noise artifacts (e.g., broadband noise or movement-related signals) result in false-positive detections exceeding ~10% of total detected events during manual review. Recordings are also considered suboptimal when signal-to-noise conditions prevent clear visual discrimination of USVs from background activity (i.e., inability to reliably distinguish USV contours from baseline noise across most of the recording). In these cases, sessions are excluded or reanalyzed following adjustment of detection parameters. Suboptimal behavioral conditioning may result in low or highly variable USV counts across sessions. Recordings producing markedly reduced USV output (e.g., &gt;50% lower total USV count relative to the cohort median at baseline for a given experimental condition) are reviewed for potential acclimation issues or procedural deviation. In these cases, total USV number and USV rate are often reduced, and extracted acoustic parameters may fall outside expected ranges or show high variability. Additional indicators of suboptimal outcomes include incomplete or inaccurate classification of USVs, often resulting from inappropriate detection settings or poor recording quality. Misclassification may be present as an overrepresentation of simple/short USV types or failure to detect multi-element (complex) vocalizations. Recordings are flagged for reanalysis when automated classification shows &lt;85% agreement with manual inspection in reviewed subsets. These issues can be identified during quality control by comparing spectrogram features with assigned classifications and by reviewing summary outputs for atypical distributions in cohort-level patterns.
Successful application of this protocol is confirmed by (1) clear visualization of USVs in spectrograms, (2) consistent detection and classification across sessions, and (3) stable or biologically interpretable variation in quantitative outcome measures. When these criteria are met, the method provides a sensitive and reproducible approach for assessing changes in vocal behavior and laryngeal function across experimental conditions.
<img alt="Spectrogram analysis of USV; raw and processed graphs of frequency vs. time for acoustic study." class="xfigimg" src="/files/ftp_upload/71661/71661fig1.jpg" data-altupdatedat="1790049645000" data-alt="Figure 1"> 
Figure 1: Example raw and processed spectrograms of representative USVs used for acoustic analysis. This figure presents unpublished pilot data from a rat aging study. Raw and processed spectrograms depict a simple (top) and complex (bottom) USV produced by a 15-month-old Sprague-Dawley female ovariectomized rat, generated using Call Classification software. X-axis = time (ms); Y-axis = frequency (kHz); color gradient = power (dB), with yellow/green indicating higher power. <a href="https://www.jove.com/files/ftp_upload/71661/71661fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Ultrasonic vocalizations analysis; bar charts showing USVs types and frequency distribution." class="xfigimg" src="/files/ftp_upload/71661/71661fig2.jpg" data-altupdatedat="1790049645000" data-alt="Figure 2"> 
Figure 2: Example USV summary outputs from a single recording session. This figure presents unpublished pilot data from a vocal training feasibility study. Representative classification results from a 10-min baseline recording of a 3-month-old transgenic male mouse overexpressing alpha-synuclein from a vocal training pilot dataset. (A) Total identified USVs categorized by classification type, demonstrating the distribution of simple and complex USVs within the session (total USVs = 42). (B) Distribution of USVs grouped by frequency modulation (ΔF, kHz), including flat (ΔF ~0), upward, downward, and complex modulation patterns. (C) Distribution of USVs grouped by total duration (Td, ms). USV detection and classification were performed using software with parameters (including detection thresholds and classification rules) defined in the Methods/Protocol section. <a href="https://www.jove.com/files/ftp_upload/71661/71661fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Graph showing statistical analysis of behavior parameters in pre/post treatment groups, OVX models." class="xfigimg" src="/files/ftp_upload/71661/71661fig3.jpg" data-altupdatedat="1790049645000" data-alt="Figure 3"> 
Figure 3: Representative USV results across Long-Evans rat hormonal and hydration manipulations. This figure is reproduced from data published in the Journal of Speech, Language, and Hearing Research37. USV measures are shown pre- and post-hydration challenge across three hormonal groups (Control; OVX + Estradiol [OVX + E2]; OVX + Placebo [OVX + PL]) and two hydration states (euhydration [EUHY]; dehydration [DEHY]) with N = 8 animals per group (age: 5–7 months). Simple (left column) and complex (right column) USV metrics are depicted: total USV number (A,B), duration (C,D), frequency range (E,F), and maximum power (G,H). Bar graphs represent group means ± SEM, and individual points depict values for each animal. Bar fill patterns indicate hormonal group: solid = Controls, slashed = OVX + E2, cross-hatched = OVX + PL. Statistical analyses were performed using generalized linear mixed-effects models (GLMMs) with Type III Wald chi-square tests to evaluate effects of hormonal group, hydration status, and their interaction. Post hoc comparisons were conducted using estimated marginal means (emmeans) with Tukey correction for multiple testing. Asterisks indicate statistically significant differences (*p &lt; .05, **p &lt; .01, ***p &lt; .001). <a href="https://www.jove.com/files/ftp_upload/71661/71661fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Vocalization analysis chart; USVs, training vs control; shows % change in vocal parameters." class="xfigimg" src="/files/ftp_upload/71661/71661fig4.jpg" data-altupdatedat="1790049645000" data-alt="Figure 4"> 
Figure 4: Representative USV results in A53T alpha-synuclein overexpression mice following a pilot vocal training paradigm. This figure presents an unpublished pilot dataset generated for a vocal training feasibility study. Data are derived from 8 mice total (age: 3–5 months), evenly distributed across groups (Control: n = 4; Training: n = 4) with balanced sex representation (two females and two males per group) and genotype distribution (Control: TG+ = 3, TG− = 1; Training: TG+ = 2, TG− = 2). USV metrics are shown as percent change from pre- to post-training for each genotype, pooled across sex. Simple (top row) and complex (bottom row) USVs are plotted separately for total USV count, duration, frequency range, and maximum intensity. Bar graphs represent group means ± SEM, and individual points depict values for each animal. Training consisted of a 5-day water-reward vocal conditioning paradigm in which mice underwent mild water restriction and were trained to emit USVs in response to water reward delivery within the recording chamber. Group differences between Control and Training conditions were assessed using two-tailed independent-samples t-tests (Welch’s correction applied when appropriate) on normalized change scores for each USV feature. No significant differences were observed between control and trained groups in this pilot dataset. <a href="https://www.jove.com/files/ftp_upload/71661/71661fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Ultrasonic vocalization (USV) software-setup.<a href="https://www.jove.com/files/ftp_upload/71661/Supplementary%20File%201.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 2: Ultrasonic vocalization (USV) software-Test recording.<a href="https://www.jove.com/files/ftp_upload/71661/Supplementary%20File%202.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 3: Ultrasonic vocalization (USV) software-Create test session.<a href="https://www.jove.com/files/ftp_upload/71661/Supplementary%20File%203.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 4: Ultrasonic vocalization (USV) software-Open test session.<a href="https://www.jove.com/files/ftp_upload/71661/Supplementary%20File%204.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 5: Ultrasonic vocalization (USV) software-Execute recording.<a href="https://www.jove.com/files/ftp_upload/71661/Supplementary%20File%205.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 6: Ultrasonic vocalization (USV) software-Finish test session.<a href="https://www.jove.com/files/ftp_upload/71661/Supplementary%20File%206.docx" target="_blank">Please click here to download this file.</a>

Laboratory rat and mouse ultrasonic vocalizations (USVs) are useful for studying vocal and neural function. This protocol describes a food-reward paradigm that reliably elicits appetitive USVs without mating-related confounds. It details animal conditioning, stimulus presentation, recording, and acoustic analysis, providing a reproducible method for studying affective-motivational and cranial sensorimotor function.

Ultrasonic vocalizations (USVs) in laboratory rats and mice provide a valuable model for investigating vocal function, affective behavior, and the ...

Our research in the Purdue Animal Behavior Core investigates cranial sensorimotor dysfunction, underlying voice and swallowing disorders, and develops standardized methods to study rat and mouse ultrasonic vocalizations. Many USV paradigms rely on mating behaviors, so this protocol uses food-reward elicitation methods to reduce social and hormonal confounds. Conduct the recordings in a darkened room or under red lighting to align with the animal's reversed light cycle.Limit room entry and remain in an adjacent space during sessions to minimize acoustic disturbances. Use sound-attenuated recording chambers to minimize external noise and electronic interference. When multiple chambers are used, assign animals to the chambers using a randomized counterbalanced schedule across recording days and experimental conditions.Record the chamber assignment for each session, including the animal identification, group, chamber number, date, and session type in a lab notebook. Clean each cage thoroughly with 70%ethanol between recordings and allow it to dry completely before the next recording. Turn on the recording computer and launch the ultrasonic vocalizations acquisition software.Click on File/Experiment, then choose New to create a new experiment. Under the Identification menu, input the experiment name. Next, in the Species menu, choose mouse.And in the Cages menu, retain the number of recording channels as four, then click on Save. Prior to each session, perform a test recording by selecting Execute Test Session, followed by Preview Ultrasounds to open the four recording channels. Click the record icon to confirm that the multichannel interface is recognized and to verify acceptable signal integrity, channel function, and noise levels.Create a test session by clicking Design Test Session, then New Test Session, and set the recording duration to 10 minutes. Assign treatment codes and animal identifiers to the appropriate chamber channels and verify session information before recording, as the settings cannot be modified afterward. To initiate the session, place the animals in the polycarbonate cages and assign them to the correct chambers.Then close the chamber door. In the recording software, click Design Test Session, then Open Test Session. Under Execute Test Session, choose Record Ultrasounds.Then click the record button to start recording and monitor the signals in real time. Allow the software to record continuously for 10 minutes and automatically stop at the end of the session. Save the recording immediately to prevent data loss by clicking the X button, then Save.Store and back up the recorded files after each session for later analysis. Remove the animals from the chambers and return them to their home cages. Immediately provide each animal with a standardized palatable food reward.Keep the reward timing consistent across sessions. Perform recording sessions once daily for three to five consecutive days following conditioning, maintaining identical timing, handling procedures and environmental conditions across all sessions. Representative recordings from a 15-month-old ovariectomized female Sprague-Dawley rat showed distinct ultrasonic vocalization contours.These included flat and frequency-modulated structures that enabled the extraction of acoustic parameters. Representative classification results from a 10-minute baseline recording of a three-month-old alpha-synuclein overexpressing transgenic male mouse demonstrates successful vocal detection with the distribution of calls across simple and complex types. It also shows distinct frequency modulation patterns, including flat, upward, downward, and complex profiles, as well as call distributions across separate duration bins.Consistent handling, conditioning, and recording conditions are essential for reliable vocalization elicitation and reproducible results. Recordings can be analyzed using alternative methods and combined with behavioral, histological, molecular, or neurophysiological analysis. Future studies can evaluate vocal changes across disease models, interventions, species, and experimental conditions.

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Research

JoVE Journal

Behavior

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild...

Hyponeophagia: A Measure of Anxiety in the Mouse

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e. lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

Studying Food Reward and Motivation in Humans

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

Pavlovian Conditioned Approach Training in Rats

Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Purdue University.  Laboratory rat and mouse ultrasonic vocalizations (USVs) are useful for studying vocal and neural function. This protocol describes a food-reward paradigm that reliably elicits appetitive USVs without mating-related confounds. It details animal conditioning, stimulus presentation, recording, and acoustic analysis, providing a reproducible method for studying affective-motivational and cranial sensorimotor function.

Anticipatory Food-Reward Elicitation of Appetitive Ultrasonic Vocalizations in Laboratory Rats and Mice

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