DOI: 10.5061/dryad.pc866t205
title: Data from: Vocal and tongue exercise in early to mid-stage Parkinson disease using the Pink1-/- rat

description: Vocal and swallowing deficits are common in Parkinson disease (PD).
 Because these impairments are resistant to dopamine replacement therapies,
 vocal and lingual exercise are the primary treatment, but not all
 individuals respond to exercise and neural mechanisms of treatment
 response are unclear. To explore putative mechanisms, we used the
 progressive Pink1-/- rat model of early to mid-stage PD and employed vocal
 and lingual exercises at 6- and 10-months of age in male Pink1-/- and wild
 type (WT) rats. We hypothesized that vocal and lingual exercise would
 improve vocal and tongue use dynamics and increase serotonin (5HT)
 immunoreactivity in related brainstem nuclei. Rats were tested at baseline
 and after 8 weeks of exercise or sham exercise. At early-stage PD (6
 months), vocal exercise resulted in increased call complexity, but did not
 change intensity, while at mid-stage (10 months), vocal exercise no longer
 influenced vocalization complexity. Lingual exercise increased tongue
 force generation and reduced relative optical density of 5HT in the
 hypoglossal nucleus at both time points. The effects of vocal and lingual
 exercise at these time points are less robust than in prodromal stages
 observed in previous work, suggesting that early exercise interventions
 may yield greater benefit. Future work targeting optimization of exercise
 at later time points may facilitate clinical translation.
description: Experimental design 
 All procedures were approved by the University of Wisconsin
 School of Medicine and Public Health Institutional Animal Care and Use
 Committee (IACUC; protocol M005177) and were conducted in accordance with
 the NIH Guide for the Care and Use of Laboratory Animals (National Research Council Committee). The following experimental design was performed with two separate cohorts of male Long-Evans rats, aged 6 months and 10 months at the time of final testing, respectively. In each cohort, three experimental groups were compared: <em>Pink1-/-</em> rats were randomized to receive vocal and lingual exercise (group 1) or sham exercise (group 2), and WT rats all received sham exercise as a control (group 3) (Table 1) (Broadfoot et al., 2023). Vocal and tongue exercise, and sham vocal and tongue exercise were completed for 8 weeks prior to time of final testing (baseline 4 months, and baseline 8 months for each age group, respectively), following standard vocal and lingual exercise protocols (Behan et al., 2012, Broadfoot et al., 2023, Schaser et al., 2012). In addition to experimental rats, 12 female Long-Evans rats were used to elicit ultrasonic vocalizations. Rats were housed in pairs in the Biomedical Research Model Services facilities of the UW School of Medicine and Public Health, were 12-hour light-cycle reversed, and underwent behavioral testing under red light during the dark period when the rats were most active. Rats were handled and weighed weekly until the exercise protocol was initiated, and throughout the remainder of the study. Standard husbandry and handling practices and procedures were used in accordance with institutional guidelines regarding animal experimentation. Table 1. Distribution of rats by condition and genotype in the 6 month and 10 month cohorts. 6 Month Cohort Condition Genotype N Exercise <em>Pink1-/-</em> 12 Sham Exercise <em>Pink1-/-</em> 12 Sham Exercise WT 12   <strong>10 Month Cohort</strong> <strong>Condition</strong> <strong>Genotype</strong> <strong>N</strong> Exercise <em>Pink1-/-</em> 14 Sham Exercise <em>Pink1-/-</em> 8 Sham Exercise WT 8 <strong>Tongue Excercise</strong> All rats were acclimated to handling and gradually water restricted to receive water freely for three hours per day. Tongue exercise was completed by <em>Pink1-/-</em> and WT rats assigned to exercise groups. Rats were trained for 2 weeks to exert at least 0.2 g of force with their tongue unto an aluminum force disk (18 mm) (Sensotec load cell, 0–250 g range) linked to a force transducer which, when pressed, immediately dispensed a 0.10 mL water reward. After two weeks of training, rats underwent baseline testing to assess their Maximal Voluntary Tongue Force (MVTF), defined as the average of the greatest 10 tongue press forces across 3 consecutive days of testing. These MVTF values were then used to establish a preset threshold for each rat. For the following eight weeks, rats completed a tongue exercise paradigm as previously described (Ciucci et al., 2011, Connor et al., 2009, Krekeler et al., 2020, Rudisch et al., 2022). Briefly, for five consecutive days per week (for all eight weeks), rats underwent five-minute exercise sessions in which each rat was required to exert a tongue press force at or above their threshold. A variable ratio (VR 5) reinforcement schedule was used to dispense water, in which anywhere from one to five tongue presses were required to receive water. After completing their exercise session, rats were given water ad libitum for three hours. <em>Pink1-/-</em> and WT rats in the non-exercise groups were also acclimated to the tongue training apparatus. They were trained to press the force transducer with the tongue to receive one water reward for demonstrating ability to complete the task, then were also provided water <em>ad libitum</em> for three hours. Following eight weeks of tongue exercise or sham exercise, final testing was completed for offline analysis. Tongue force and timing behaviors testing & analysis The MVTF data point in millinewtons (mN) were calculated from a three-day average of the highest press force during tongue exercise sessions (see above). We also assessed changes in the variability of force generation by calculating the coefficient of variation for each rat at the baseline and final time points. On an exploratory basis, we investigated changes to press force over the course of a testing session by assessing press force at 30-second time intervals (Broadfoot et al., 2023, Glass et al., 2019). <strong>Vocal Excercise</strong> On each exercise day, vocal exercise was completed following tongue exercise. A well-established, reliable social mating paradigm was used to elicit male vocalizations (Grant et al., 2015, Kelm-Nelson et al., 2015, Ringel et al., 2013). Briefly, this paradigm introduces a sexually receptive female into a male rat’s home cage, allowing the male rat to show interest (<em>i.e.</em>, via mounting, chasing, or sniffing). Estrus is confirmed by observing a combination of behavioral signs (<em>e.g.</em>, lordosis, ear wiggling, darting, and hopping). The female is left in the male rat’s home cage for up to five minutes, and removed after prolonged approach behaviors during the initiation stage for up to 5 min (<em>i.e.</em>, chasing, sniffing, autogrooming), or after two mounting attempts by the male rat, and then is removed. All male rats in both genotypes displayed a variety of expected behaviors after removal of the stimulus rat, as well as a variety of call types. All rats, irrespective of their assigned group, were acclimated to this paradigm for two weeks. After two weeks of acclimation, <em>Pink1-/-</em> rats assigned to the exercise group underwent a vocal exercise protocol five days per week for eight weeks. After removal of the female, male 50-kilohertz (kHz) frequency modulated (FM) vocalizations were tracked. When these vocalizations were produced, a pen-click sound was made and a highly palatable food reward was administered (Kelm-Nelson et al., 2015) until the rat produced 30 strings of target vocalizations and therefore received 30 rewards. A classic method of reinforcement for successive approximation was used to reward the production of calls with increasing complexity and loudness as the vocal exercise period progressed. <em>Pink1-/-</em> and WT rats that were in the sham exercise groups underwent behavioral reinforcement procedures but without vocal exercise. Instead, rats were trained to receive a pen click and food reward for moving to an assigned corner of the testing cage. Following eight weeks of vocal or sham exercise, final testing was completed. USVs were recorded for 90 seconds (s) per experimental rat after the female was removed. Recordings were taken at baseline and final time points for both the 6-month and 10-month cohorts. Recordings were obtained with an ultrasonic microphone with 16-bit resolution and sampling rate of 250 kHz (CM16, Avisoft Bioacoustics, Berlin, Germany), which was mounted 15 cm above the male rat’s standard polycarbonate cage. This set-up was paired with <em>Avisoft</em> software (Avisoft Bioacoustics, Berlin, Germany) for recording and subsequent analysis. <strong>USV analysis</strong> Recorded vocalizations from testing days were analyzed offline in <em>Avisoft</em> (Avisoft Bioacoustics, Berlin, Germany). Acoustic analysis involved the creation of a spectrogram per waveform using: Fast Fourier Transformation (FFT; 512 points, frame size of 100 %, and temporal resolution of 75 % overlap), and a high pass filter (noise reduction below 25-kilohertz (kHz). The rate of vocalizations was decreased by a factor of 10, allowing calls to 1) be audible to the human ear and 2) be categorized into call types. Calls were identified and labeled with the following call type categories: simple, simple compound, frequency modulated (FM), FM compound, harmonic, and harmonic compound. Additionally, complex calls were operationally defined as all non-simple calls (<em>i.e.</em>, simple compound, FM, FM compound, harmonic, and harmonic compound). Analysis of acoustic measures was completed using SASLab Pro, a statistical program which provided the averages of the following outcomes: call rate (calls/second), intensity of the call (decibel-dB), duration of the call (milliseconds-ms), and peak frequency (kHz) of the call. This methodology has been validated in prior research (Grant et al., 2015, Ciucci et al., 2007, Wright et al., 2010, Pultorak et al., 2016). <strong>Tissue Processing</strong> After behavioral testing at the final time points was completed (6 and 10 months of age), rats were deeply anesthetized using isoflurane, and underwent transcardial perfusion. Perfusion included an initial injection of 200 mL of cold saline, followed by a 500 mL injection of paraformaldehyde (4 %) combined with a phosphate-buffered solution (1 % – PBS). Immediately following perfusion, brains were removed and fixed in 4 % paraformaldehyde at 4 °C. Following an overnight incubation, brains were transferred and stored at 4 °C in 0.02 % sodium azide in 0.1 M PBS solution prior to sectioning. When scheduled for sectioning, brains were removed from the solution and incubated in sucrose at 4 °C overnight. <strong>Tissue Slicing</strong> Cryoprotectant solution was prepared ahead of scheduled sectioning. To prepare for sectioning, brains were affixed on a stage perpendicular to the floor and encapsulated with Tissue Tek OCT compound (Kura) and allowed to come to temperature (∼-12 °C) for approximately 20 min. Tissue was then sliced through the cortex and brainstem in a coronal direction at 40-µm or 50-µm using a Leica® CM 1850 Cryostat. Brain slices were stored in 12- or 24-well plates, free-floating, in cryoprotectant at −20 °C until completion of histological assays, in line with prior methodology (Broadfoot et al., 2023). <strong>Immunohistochemistry</strong> Brainstem tissue reserved for 5-HT staining was processed as previously described (Grant et al., 2015, Johnson et al., 2020). In brief, slices underwent a series of brief washes in 0.1 M PBS, were incubated in 0.5 % H2O2 and 0.1 M PBS solution, and were washed in 0.1 M PBS and PBS-T. 20 % normal goat serum with PBS-T was used to block tissue for 1 h. Following blocking, tissue was incubated in primary antibody (1:10,000 5-HT, Immunostar Product ID 20080) and PBS-T overnight at 4 °C. The following day, tissue was washed in PBS-T, then incubated in secondary antibody (1:500 concentration of conjugated biotinylated goat anti-rabbit, Vector Laboratories, BA1000) at room temperature for 2 h. After incubation, tissue was washed in PBS-T, then AB solution (VECTASTAIN® Elite ABC-HRP Kit, Peroxidase (Goat IgG)) was applied for 1 h to tag secondary antibody. Following washes in 0.1 M PBS, tissue was submerged into SIGMAFAST 3,3-diaminobenzidine (DAB; Sigma Aldrich, St. Louis, MO) for visualization. In preparation for microscopy, neural tissue slices were float-mounted onto gelatin-coated slides, dehydrated in a series of alcohols and xylenes, and cover slipped using Cytoseal (Cytoseal™ Mounting Medium, Richard-Allan Scientific). <strong>Microscopy and relative optical density analysis</strong> Following 5-HT staining, slides were imaged using an Olympus BX53 Upright Microscope prior to completion of relative optical density analysis. This method of quantification was chosen to account for potential variation in staining across 40-µm or 50-µm thick sections. To conduct relative optical density analysis of immunoreactivity of 5-HT in the hypoglossal nucleus and raphe obscurus, bilateral sections containing one or both regions were visualized at 4x magnification using the Slide Scanning Workflow in Stereo Investigator <em>®</em> (mean number of hypoglossal nucleus sections = 4.1, mean number of raphe obscurus sections = 5.5) Slices were then traced, background image was corrected, and tissue was imaged at 10x magnification using the Slide Scanning Workflow in Stereo Investigator <em>®</em>. Relative optical density analysis of hypoglossal nuclei (Bregma range from −12.72 to −14.76) and raphe obscurus (Bregma range −11.64 to −14.28) were completed using ImageJ (Rasband) (U.S. National Institutes of Health, Bethesda, MD). To conduct the analysis, RBG color images were converted to grayscale (8-bit), and a 350 x 350 pixel box was placed within the region of interest (hypoglossal nucleus (bilateral) or raphe obscurus (center), respectively) for each slice imaged. Additionally, a 50 x 50 pixel box was drawn in a region where staining was absent from each slice imaged; an optical density reading was done for the selected regions. To calculate relative optical density, the following calculation was completed:ROD=averageODfromROIofeachhemisphere-ODfromregiondevoidofstainThe resulting number was then averaged for each rat, per region of interest, across sections.   <strong>Statistical Analysis</strong>   Two-way repeated measures analyses of variance (ANOVA) were performed separately for each age cohort to assess interactions between treatment condition and time point (baseline prior to training, and final after training). One-way ANOVA were performed separately for each age cohort to assess the effect of treatment condition on the relative optical density of 5-HT in the hypoglossal nucleus and the raphe obscurus. Non-normally distributed data were analyzed with Kruskal Wallis tests. Significance level was set <em>a priori</em> at 0.05. Post-hoc testing was performed with Holm-Sidak correction for multiple comparisons for repeated measures ANOVA, and Bonferroni correction for multiple comparisons for Kruskal-Wallis tests. All statistical analysis was completed with R statistical software, version 4.2.1.
description: # Vocal and Tongue Exercise in Early to Mid-stage Parkinson Disease using
 the Pink1-/- Rat: Open Source Data  **Description of the data, software,
 and file structure**   We have uploaded the relative optical density
 averages for the 6month old animals
 (*6mo_RoD_Averages_Master_Sheet_IDs_Removed.xlsx*), the relative optical
 density averages for the 10 month old animals
 (*10mo_RoD_Averages_Master_Sheet_IDs_Removed.xlsx*), the USV statistical
 data for the 6 month old animals (*NP_6mo_USV_IDs_Removed.xlsx*), the USV
 statistical data for the 10 month old animals
 (*NP_USV_10mo_IDs_Removed.xlsx*), the tongue force data for both the 6 and
 10 month old animals (*NP6_10Tongue_IDs_Removed.xlsx*), and the protocol
 used for collecting the relative optical density
 (*Optical_Density_Procedure_1.28.2022.docx*). **Abbreviations** WT- Wild
 type RoD- Relative Optical Density 12N- Hypoglossal Nucleus Rob- Nucleus
 Raphe Obscurus N/A- Not applicable, used to fill cells that were
 missing/did not have data for them. Software_Descriptions 1\)Image J    
 This program was used to process and analyze the captured microscopic
 images of the stained brain tissues. 2\)DeepSqueak     This program was
 used to analyze, characterize and draw values for statistical analysis
 from calls collected using and ultrasonic microphone. This program is
 accessed through the MatLab program. File_Description
 1\)**6mo_RoD_Averages_Master_Sheet_IDs_Removed.xlsx**    **ID**-
 Randomized number assigned to identify an animal. Removed to de-identify
 the data.    **12N_RoD_Average**- The average relative optical density
 found in the hypoglossal nucleus.    **Rob_RoD_Average**- The average
 relative optical density found in the nucleus raphe obscurus.  
  **Condition**-         Pink_Exercise- Pink1-/- animals that underwent
 exercise conditions.         Pink_Non- Pink1-/- animal that did not
 ungerdo any exericise conditions.         WT_Control- Wild type animal
 used as a control. 2\)**10mo_RoD_Averages_Master_Sheet_IDs_Removed.xlsx**
     **ID**- Randomized number assigned to identify an animal.    
 **12N_RoD_Averages**- The average relative optical density found in the
 hypoglossal nucleus.     **Rob_RoD_Averages**- The average relative
 optical density found in the nucleus raphe obscurus.     **Condition**-  
       Pink_Exercise- Pink1-/- animals that underwent exercise conditions.
         Pink_Sham- Pink1-/- animal that did not undergo any exercise
 conditions.         WT_Sham- Wild Type animal used as a control.
 3\)**NP_6mo_USV_IDs_Removed.xlsx**     **ID**- Randomized number assigned
 to identify an animal. Removed to de-identify the data.    
 **Timpepoint**-         Baseline- The initial testing day.         Final-
 Final testing day.     **Condition**-         Pink_non- Pink1 knockout
 animals that did not undergo exercise conditions.         Pink_exercise-
 Pink1 knockout animals that underwent exercise conditions.         WT-
 Wild type animals used as a control.     **Total_Calls_All**- The total
 amount of calls the animal made.     **Average_Duration_All**- The average
 duration of calls measured in miliseconds.     **Max_Duration_All**- The
 max duration for all calls made.     **Top_10 \_Duration_All**- The
 average of the top 10 calls with the longest duration for all calls made.
     **Average \_Bandwidth_All**- The average bandwidth for all calls the
 animal made. Measured in kHz.     **Max_Bandwidth_All**- The maximum
 bandwidth of all calls the animal made. Measured in kHz.    
 **Top_10_Bandwidth_All**- The average bandwidth of the top 10 of all calls
 the animals made. Measured in kHz.     **Average_Intensity_All**- The
 average intensity of all calls made.     **Max_Intensity_All**- The
 maximum intensity of all calls the animal made.    
 **Top_10-Intensity_All**- The average intenstity of the top 10 of all
 calls the animal made.     **Average_Peak_Frequency_All**- The average
 peak frequency of all calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_All**- The maximum peak frequency of all the calls
 the animal made. Measured in kHz.     **Top_10_Peak_Frequency_All**- The
 average of the top 10 peak frequencies of all the calls the animal made.  
   **Percent_Complex_All**- The percentage of calls the animal made that
 fell into the complex category.     **Range_of_Intensity_All**- The range
 of intensity of all calls the animal made.     **Call_Rate_All**- The rate
 at which the animal called for all calls made.     **Total_Calls_Simple**-
 The total amount of simple calls the animal made.    
 **Average_Duration_Simple**- The average duration of simple calls the
 animal made measured in miliseconds.     **Max_Duration_Simple**- The max
 duration for all simple calls made.     **Top_10_Duration_Simple**- The
 average of the top 10 calls with the longest duration for all simple calls
 made.     **Average_Bandwidth_Simple**- The average bandwidth for all
 simple calls the animal made. Measured in kHz.    
 **Max_Bandwidth_Simple**- The maximum bandwidth of all simple calls the
 animal made. Measured in kHz.     **Top_10_Bandwidth_Simple**- The average
 bandwidth of the top 10 of all simple calls the animals made. Measured in
 kHz.     **Average_Intensity_Simple**- The average intensity of all simple
 calls made.     **Max_Intensity_Simple**- The maximum intensity of all
 simple calls the animal made.     **Top_10_Intensity_Simple**- The average
 intenstity of the top 10 of all simple calls the animal made.    
 **Average_Peak_Frequency_Simple**- The average peak frequency of all
 simple calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_Simple**- The maximum peak frequency of all simple
 calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_Simple**- The average of the top 10 peak
 frequencies of all simple calls the animal made. Measured in kHz.    
 **Percent_Complex_Simple**- The percentage of simple calls the animal made
 that fell into the complex category.     **Range_of_Intensity_Simple**-
 The range of intensity of all simple calls the animal made.    
 **Call_Rate_Simple**- The rate at which the animal called for all calls
 made.     **Total_Calls_Simple_cmpd**- The total amount of simple compound
 calls the animal made.     **Average_Duration_Simple_cmpd**- The average
 duration of simple compound calls the animalmade measured in miliseconds.
     **Max_Duration_Simple_cmpd**- The max duration for all simple compound
 calls made.     **Top_10_Duration_Simple_cmpd**- The average of the top 10
 calls with the longest duration for all simple compound calls made.    
 **Average_Bandwidth_Simple_cmpd**- The average bandwidth for all simple
 compound calls the animal made. Measured in kHz.    
 **Max_Bandwidth_Simple_cmpd**- The maximum bandwidth of all simple
 compound calls the animal made. Measured in kHz.    
 **Top_10_Bandwidth_Simple_cmpd**- The average bandwidth of the top 10 of
 all simple compound calls the animals made. Measured in kHz.    
 **Average_Intensity_Simple_cmpd**- The average intensity of all simple
 compound calls made.     **Max_Intensity_Simple_cmpd**- The maximum
 intensity of all simple compound calls the animal made.    
 **Top_10_Intensity_Simple_cmpd**- The average intenstity of the top 10 of
 all simple compound calls the animal made.    
 **Average_Peak_Freq_Simple_cmpd**- The average peak frequency of all
 simple compound calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_Simple_cmpd**- The maximum peak frequency of all
 simple compound calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_Simple_cmpd**- The average of the top 10 peak
 frequency simple compound calls the animal made. Measured in kHz.    
 **Percent_Complex_Simple_cmpd**- The percentage of simple compound calls
 the animal made that fell into the complex category.    
 **Range_of_Intensity_Simple_cmpd**- The range of intensity of all simple
 compound calls the animal made.     **Call_Rate_Simple_cmpd**- The rate at
 which the animal called for all simple compound calls made.    
 **Total_Calls_Harmonic**- The total amount of harmonic calls the animal
 made.     **Average_Duration_Harmonic**- The average duration of harmonic
 calls in miliseconds.     **Max_Duration_Harmonic**- The max duration for
 all harmonic calls made.     **Top_10_Duration_Harmonic**- The average of
 the top 10 calls with the longest duration for all harmonic calls made.  
   **Average_Bandwidth_Harmonic**- The average bandwidth for all harmonic
 calls the animal made. Measured in kHz.     **Max_Bandiwdth_Harmonic**-
 The maximum bandwidth of all harmonic calls the animal made. Measured in
 kHz.     **Top_10_Bandwidth_Harmonic**- The average bandwidth of the top
 10 of all harmonic calls the animals made. Measured in kHz.    
 **Average_Intensity_Harmonic**- The average intensity of all harmonic
 calls made.     **Max_Intensity_Harmonic**- The maximum intensity of all
 harmonic calls the animal made.     **Top_10_Intensity_Harmonic**- The
 average intenstity of the top 10 of all harmonic calls the animal made.  
   **Average_Peak_Frequency_Harmonic**- The average peak frequency of all
 harmonic calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_Harmonic**- The maximum peak frequency of all
 harmonic calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_Harmonic**- The average of the top 10 peak
 frequencies of all harmonic calls the animal made. Measured in kHz.    
 **Percent_Complex_Harmonic**- The percentage of harmonoic calls the animal
 made that fell into the complex category.    
 **Range_of_Intensity_Harmonic**- The range of intensity of all harmonic
 calls the animal made.     **Call_Rate_Harmonic**- The rate at which the
 animal called for all harmonic calls made.     **Total_Calls_FM**- The
 total amount of frequency modulated calls the animal made.    
 **Average_Duration_FM**- The average duration of frequency modulated calls
 in miliseconds.     **Max_Duration_FM**- The max duration for all
 frequency modulated calls made.     **Top_10_Duration_FM**- The average of
 the top 10 calls with the longest duration for all frequency modulated
 calls made.     **Average_Bandwidth_FM**- The average bandwidth for all
 frequency modulated calls the animal made. Measured in kHz.    
 **Max_Bandiwdth_FM**- The maximum bandwidth of all frequency modulated
 calls the animal made. Measured in kHz.     **Top_10_Bandwidth_FM**- The
 average bandwidth of the top 10 of all frequency modulated calls the
 animals made. Measured in kHz.     **Average_Intensity_FM**- The average
 intensity of all frequency modulated calls made.     **Max_Intensity_FM**-
 The maximum intensity of all frequency modualted calls the animal made.  
   **Top_10_Intensity_FM**- The average intenstity of the top 10 of all
 frequency modulated calls the animal made.    
 **Average_Peak_Frequency_FM**- The average peak frequency of all frequency
 modulated calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_FM**- The maximum peak frequency of all frequency
 modulated calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_FM**- The average of the top 10 peak frequencies
 of all frequency modulated calls the animal made. Measured in kHz.    
 **Percent_Complex_FM**- The percentage of frequency modulated calls the
 animal made that fell into the complex category.    
 **Range_of_Intensity_FM**- The range of intensity of all frequency
 modulated calls the animal made.     **Call_Rate_FM**- The rate at which
 the animal called for all frequency modulated calls made.    
 **Proportion_of_Calls_Simple**- The proportion of all calls made that were
 categorized as simple.     **Proportion_of_Calls_Simple_cmpd**- The
 proportion of all calls made that were categorized as simple compound.    
 **Proportion_of_Calls_Harmonic**- The proportion of all calls made that
 were categorized as harmonic.     **Proportion_of_Calls_FM**- The
 proportion of all calls made that were categorized as frequency modulated.
 4\)**NP_USV_10mo_IDs_Removed.xlsx**     **ID**- Randomized number assigned
 to identify an animal. Removed to de-identify the data.    
 **Timpepoint**-         Baseline- The initial testing day.         Final-
 Final testing day.     **Condition**-         Pink_non- Pink1 knockout
 animals that did not undergo exercise conditions.         Pink_exercise-
 Pink1 knockout animals that underwent exercise conditions.         WT-
 Wildetype animals used as a control.     **Total_Calls_All**- The total
 amount of calls the animal made.     **Average_Duration_All**- The average
 duration of calls measured in miliseconds.     **Max_Duration_All**- The
 max duration for all calls made.     **Top_10 \_Duration_All**- The
 average of the top 10 calls with the longest duration for all calls made.
     **Average \_Bandwidth_All**- The average bandwidth for all calls the
 animal made. Measured in kHz.     **Max_Bandwidth_All**- The maximum
 bandwidth of all calls the animal made. Measured in kHz.    
 **Top_10_Bandwidth_All**- The average bandwidth of the top 10 of all calls
 the animals made. Measured in kHz.     **Average_Intensity_All**- The
 average intensity of all calls made.     **Max_Intensity_All**- The
 maximum intensity of all calls the animal made.    
 **Top_10-Intensity_All**- The average intenstity of the top 10 of all
 calls the animal made.     **Average_Peak_Frequency_All**- The average
 peak frequency of all calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_All**- The maximum peak frequency of all the calls
 the animal made. Measured in kHz.     **Top_10_Peak_Frequency_All**- The
 average of the top 10 peak frequencies of all the calls the animal made.
 Measured in kHz.     **Percent_Complex_All**- The percentage of calls the
 animal made that fell into the complex category.    
 **Range_of_Intensity_All**- The range of intensity of all calls the animal
 made.     **Call_Rate_All**- The rate at which the animal called for all
 calls made.     **Total_Calls_Simple**- The total amount of simple calls
 the animal made.     **Average_Duration_Simple**- The average duration of
 simple calls the animal made measured in miliseconds.    
 **Max_Duration_Simple**- The max duration for all simple calls made.    
 **Top_10_Duration_Simple**- The average of the top 10 calls with the
 longest duration for all simple calls made.    
 **Average_Bandwidth_Simple**- The average bandwidth for all simple calls
 the animal made. Measured in kHz.     **Max_Bandwidth_Simple**- The
 maximum bandwidth of all simple calls the animal made. Measured in kHz.  
   **Top_10_Bandwidth_Simple**- The average bandwidth of the top 10 of all
 simple calls the animals made. Measured in kHz.    
 **Average_Intensity_Simple**- The average intensity of all simple calls
 made.     **Max_Intensity_Simple**- The maximum intensity of all simple
 calls the animal made.     **Top_10_Intensity_Simple**- The average
 intenstity of the top 10 of all simple calls the animal made.    
 **Average_Peak_Frequency_Simple**- The average peak frequency of all
 simple calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_Simple**- The maximum peak frequency of all simple
 calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_Simple**- The average of the top 10 peak
 frequencies of all simple calls the animal made. Measured in kHz.    
 **Percent_Complex_Simple**- The percentage of simple calls the animal made
 that fell into the complex category.     **Range_of_Intensity_Simple**-
 The range of intensity of all simple calls the animal made.    
 **Call_Rate_Simple**- The rate at which the animal called for all calls
 made.     **Total_Calls_Simple_cmpd**- The total amount of simple compound
 calls the animal made.     **Average_Duration_Simple_cmpd**- The average
 duration of simple compound calls the animalmade measured in miliseconds.
     **Max_Duration_Simple_cmpd**- The max duration for all simple compound
 calls made.     **Top_10_Duration_Simple_cmpd**- The average of the top 10
 calls with the longest duration for all simple compound calls made.    
 **Average_Bandwidth_Simple_cmpd**- The average bandwidth for all simple
 compound calls the animal made. Measured in kHz.    
 **Max_Bandwidth_Simple_cmpd**- The maximum bandwidth of all simple
 compound calls the animal made. Measured in kHz.    
 **Top_10_Bandwidth_Simple_cmpd**- The average bandwidth of the top 10 of
 all simple compound calls the animals made. Measured in kHz.    
 **Average_Intensity_Simple_cmpd**- The average intensity of all simple
 compound calls made.     **Max_Intensity_Simple_cmpd**- The maximum
 intensity of all simple compound calls the animal made.    
 **Top_10_Intensity_Simple_cmpd**- The average intenstity of the top 10 of
 all simple compound calls the animal made.    
 **Average_Peak_Freq_Simple_cmpd**- The average peak frequency of all
 simple compound calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_Simple_cmpd**- The maximum peak frequency of all
 simple compound calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_Simple_cmpd**- The average of the top 10 peak
 frequency simple compound calls the animal made. Measured in kHz.    
 **Percent_Complex_Simple_cmpd**- The percentage of simple compound calls
 the animal made that fell into the complex category.    
 **Range_of_Intensity_Simple_cmpd**- The range of intensity of all simple
 compound calls the animal made.     **Call_Rate_Simple_cmpd**- The rate at
 which the animal called for all simple compound calls made.    
 **Total_Calls_Harmonic**- The total amount of harmonic calls the animal
 made.     **Average_Duration_Harmonic**- The average duration of harmonic
 calls in miliseconds.     **Max_Duration_Harmonic**- The max duration for
 all harmonic calls made.     **Top_10_Duration_Harmonic**- The average of
 the top 10 calls with the longest duration for all harmonic calls made.  
   **Average_Bandwidth_Harmonic**- The average bandwidth for all harmonic
 calls the animal made. Measured in kHz.     **Max_Bandiwdth_Harmonic**-
 The maximum bandwidth of all harmonic calls the animal made. Measured in
 kHz.     **Top_10_Bandwidth_Harmonic**- The average bandwidth of the top
 10 of all harmonic calls the animals made. Measured in kHz.    
 **Average_Intensity_Harmonic**- The average intensity of all harmonic
 calls made.     **Max_Intensity_Harmonic**- The maximum intensity of all
 harmonic calls the animal made.     **Top_10_Intensity_Harmonic**- The
 average intenstity of the top 10 of all harmonic calls the animal made.  
   **Average_Peak_Frequency_Harmonic**- The average peak frequency of all
 harmonic calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_Harmonic**- The maximum peak frequency of all
 harmonic calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_Harmonic**- The average of the top 10 peak
 frequencies of all harmonic calls the animal made. Measured in kHz.    
 **Percent_Complex_Harmonic**- The percentage of harmonoic calls the animal
 made that fell into the complex category.    
 **Range_of_Intensity_Harmonic**- The range of intensity of all harmonic
 calls the animal made.     **Call_Rate_Harmonic**- The rate at which the
 animal called for all harmonic calls made.     **Total_Calls_FM**- The
 total amount of frequency modulated calls the animal made.    
 **Average_Duration_FM**- The average duration of frequency modulated calls
 in miliseconds.     **Max_Duration_FM**- The max duration for all
 frequency modulated calls made.     **Top_10_Duration_FM**- The average of
 the top 10 calls with the longest duration for all frequency modulated
 calls made.     **Average_Bandwidth_FM**- The average bandwidth for all
 frequency modulated calls the animal made. Measured in kHz.    
 **Max_Bandiwdth_FM**- The maximum bandwidth of all frequency modulated
 calls the animal made. Measured in kHz.     **Top_10_Bandwidth_FM**- The
 average bandwidth of the top 10 of all frequency modulated calls the
 animals made. Measured in kHz.     **Average_Intensity_FM**- The average
 intensity of all frequency modulated calls made.     **Max_Intensity_FM**-
 The maximum intensity of all frequency modualted calls the animal made.  
   **Top_10_Intensity_FM**- The average intenstity of the top 10 of all
 frequency modulated calls the animal made.    
 **Average_Peak_Frequency_FM**- The average peak frequency of all frequency
 modulated calls the animal made. Measured in kHz.    
 **Max_Peak_Frequency_FM**- The maximum peak frequency of all frequency
 modulated calls the animal made. Measured in kHz.    
 **Top_10_Peak_Frequency_FM**- The average of the top 10 peak frequencies
 of all frequency modulated calls the animal made. Measured in kHz.    
 **Percent_Complex_FM**- The percentage of frequency modulated calls the
 animal made that fell into the complex category.    
 **Range_of_Intensity_FM**- The range of intensity of all frequency
 modulated calls the animal made.     **Call_Rate_FM**- The rate at which
 the animal called for all frequency modulated calls made.    
 **Proportion_of_Calls_Simple**- The proportion of all calls made that were
 categorized as simple.     **Proportion_of_Calls_Simple_cmpd**- The
 proportion of all calls made that were categorized as simple compound.    
 **Proportion_of_Calls_Harmonic**- The proportion of all calls made that
 were categorized as harmonic.     **Proportion_of_Calls_FM**- The
 proportion of all calls made that were categorized as frequency modulated.
 5\)**NP6_10Tongue_IDs_Removed.xlsx**     **Group**- Randomly assigned
 group number     **Timepoint**-         Baseline- Initail testing day    
     Final- Final testing day     **Animal_ID**- Randomized number assigned
 to identify an animal. Removed to de-identify the data.     **Max_Press**-
 Highest pressed value in grams     **Max_Press_mN**- Highest pressed value
 in milinewtons     **Avg_Top_Ten**- Average of 10 highest presses    
 **SD_Top_Ten**- Standard deviation for the 10 highest presses    
 **Avg_All**- Average press force for whole session     **SD_All**-
 Standard deviation for all tongue presses.    
 **Coefficient_of_Variation**- Statistical measure to express the relative
 dispersion of data points around the mean.     **Weight**- Weight of the
 animal     **Genotype**-         Pink1- Animal that is a Pink1 gene
 knockout         WT- Standard wildtype rat     **End_Point**- Age of
 animal in months at time of Final collection timepoint     **Treatment**-
         Pink_Exercise- Pink1 knockout animals that underwent exercise
 conditions         Pink_non- Pink1 knockout animals that did not undergo
 exercise conditions         WT- Wildetype animals used as a control    
 **Pressrate30**- Number of presses from 0-30 seconds     **Pressrate60**-
 Number of presses from 31-60 seconds     **Pressrate90**- Number of
 presses from 61-90 seconds     **Pressrate120**- Number of presses from
 91-120 seconds     **Pressrate150**- Number of presses from 121-150
 seconds     **Pressrate180**- Number of presses from 151-180 seconds    
 **Pressrate210**- Number of presses from 181-210 seconds    
 **Pressrate240**- Number of presses from 211-240 seconds    
 **Pressrate270**- Number of presses from 241-270 seconds    
 **Pressrate300**- Number of presses from 271-300 seconds    
 **Pressure30**- Amount of pressure in milinewtons from 0-30 seconds    
 **Pressure60**- Amount of pressure in milinewtons from 31-60 seconds    
 **Pressure90**- Amount of pressure in milinewtons from 61-90 seconds    
 **Pressure120**- Amount of pressure in milinewtons from 91-120 seconds    
 **Pressure150**- Amount of pressure in milinewtons from 121-150 seconds  
   **Pressure180**- Amount of pressure in milinewtons from 151-180 seconds
     **Pressure210**- Amount of pressure in milinewtons from 181-210
 seconds     **Pressure240**- Amount of pressure in milinewtons from
 211-240 seconds     **Pressure270**- Amount of pressure in milinewtons
 from 241-270 seconds     **Pressure300**- Amount of pressure in
 milinewtons from 271-300 seconds  
 6\)**Optical_Density_Procedure_1.28.2022.docx**     Word document (.docx)
 that contains a walkthrough of the process and steps used to evaluate the
 tissue collected to find the relative optical density values.