Vocal and tongue exercise in early to mid-stage Parkinson disease using the Pink1-/- rat

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.

Methods

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: Pink1-/- 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
Pink1-/-
12

Sham Exercise
Pink1-/-
12

Sham Exercise
WT
12

10 Month Cohort

Condition
Genotype
N

Exercise
Pink1-/-
14

Sham Exercise
Pink1-/-
8

Sham Exercise
WT
8

Tongue Excercise

All rats were acclimated to handling and gradually water restricted to receive water freely for three hours per day. Tongue exercise was completed by Pink1-/- 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. Pink1-/- 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 ad libitum 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).

Vocal Excercise

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 (i.e., via mounting, chasing, or sniffing). Estrus is confirmed by observing a combination of behavioral signs (e.g., 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 (i.e., 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, Pink1-/- 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. Pink1-/- 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 Avisoft software (Avisoft Bioacoustics, Berlin, Germany) for recording and subsequent analysis.

USV analysis

Recorded vocalizations from testing days were analyzed offline in Avisoft (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 (i.e., 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).

Tissue Processing

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.

Tissue Slicing

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).

Immunohistochemistry

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).

Microscopy and relative optical density analysis

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 ® (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 ®. 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.

Statistical Analysis

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 a priori 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.