Vocal and Tongue Exercise in early to mid-stage Parkinson disease using the Pink1−/− Rat

Abstract

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.

1: Introduction

Vocal deficits (hypokinetic dysarthria) and swallowing deficits occur in up to 90% and 87% of individuals with Parkinson disease (PD), respectively^1–8. Both have a substantial negative impact on quality of life^9–15. Swallowing deficits are also associated with aspiration pneumonia^16–19, the primary cause of death in PD. Unlike limb motor deficits, vocal deficits and swallowing deficits respond minimally to dopamine replacement^20–25, suggesting that their disease-specific neurological mechanisms are at least partially extradopaminergic^26,27. In particular, disruption of brainstem serotonergic (5HT) and noradrenergic systems involving cranial sensory and motor nuclei are likely components of vocal and lingual deficits in PD, with a number of studies demonstrating brainstem 5HT and noradrenergic disruption prior to onset of classical limb motor symptoms^28–33. The hypoglossal nucleus receives 5HT input from a number of medullary nuclei including the raphe obscurus, and hypoglossal motoneurons innervate tongue musculature important for swallow function. Importantly, tongue strengthening exercise can reverse age-related degradation of 5HT input to the hypoglossal nucleus³⁴, and serotonergic manipulation has a strong influence on vocalization³³, making 5HT disruption in the hypoglossal nucleus a particularly promising target for investigation of cranial sensorimotor deficits in PD, and for understanding and optimizing voice and swallowing treatment.

Because vocal deficits and swallowing deficits do not respond to standard pharmacologic treatment^35,36, voice and swallow exercise are often prescribed. Voice exercise in PD is targeted at improvement of vocal fold adduction, and improvement of respiratory-phonatory coordination, with patients instructed to produce a loud voice with high effort during various speech tasks and sustained vowels^37,38. Because reduced tongue strength is an important contributor to functional swallowing deficits in PD³⁹, swallow exercises typically aim to improve tongue strength and endurance^40–42. While a degree of symptomatic improvement is often observed following exercise, this effect is not universal and decreases with disease progression^38,43–45. There are many factors that could influence the outcomes of vocal and swallow exercise, such as: the stage in the disease process at which exercise is initiated; the dose and duration of exercise; the effects of concomitant versus separated courses of vocal and swallow exercise; the interactions among pharmacologic interventions and exercise. In humans, we cannot predict the exact time of disease onset nor the rate of disease progression, and thus do not have sufficient experimental control to manipulate and study factors that could improve efficacy of voice and swallow exercise in this patient population. Further, logistical challenges with analyzing human neural tissue early in the disease process prevent the systematic investigation of extradopaminergic elements that might cause vocal and swallowing deficits.

In the absence of sufficient experimental control to thoroughly investigate voice and swallowing deficits of humans, the Pink1−/− rat model of voice and swallowing deficits in PD has been used to study voice and swallowing deficit pathophysiology, and is a useful tool for studying optimization of the response of these deficits to treatment^36,46–48. The Pink1−/− rat model is based on a form of familial, early onset PD (PARK6). This progressive PD model has been shown to exhibit deficits in vocalization, swallowing, and other “non-motor” signs of PD^28,49–52. Further, vocal and swallowing deficits in Pink1−/− rats have been shown to be responsive to exercise and noradrenergic manipulation (as opposed to standard dopamine replacement therapies)^53–55.

It has also been demonstrated that vocal and swallowing deficits occur prior to the onset of motor deficits in the Pink1−/− rat, similar to human PD⁵⁶. This provides a means for studying not only the neurobiological mechanisms of disease progression, but for systematically assessing factors that can influence outcomes of exercise-based interventions, such as stage of disease progression. Recently, Broadfoot and colleagues⁵⁷ observed improvements in vocalization and tongue function in Pink1−/− rats undergoing vocal and tongue exercise at 4 months of age. While the findings are promising, the 4-month time point corresponds to a prodromal stage of disease progression⁵⁶, prior to the point at which most patients with PD would be diagnosed and would receive vocal and swallowing exercise. Accordingly, it is clinically relevant to examine the effects of exercise on vocal and swallowing function outcomes at later stages of disease progression and central extradopaminergic disruption.

The primary goal of this study was to determine the effects of concurrent vocal and tongue exercise on vocalization and tongue function at 6 and 10 months of age in the Pink1−/− rat model of PD. These time points correspond to stages of disease progression that are more likely to represent the time at which vocal and tongue exercise commences in patients with early-mid stage PD. The secondary goal of this study was to explore the responsiveness of extradopaminergic systems to this exercise program. We hypothesized that, at both 6 months and 10 months, rats undergoing vocal and swallowing exercise would demonstrate improved vocal and swallowing outcomes, and increased serotonergic immunoreactivity in the raphe obscurus and hypoglossal nucleus.

2: Methods

2.1: 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 Animals⁵⁸. 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)⁵⁷. 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^34,57,59. 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	Pinkl−/−	12
Sham Exercise	Pinkl−/−	12
Sham Exercise	WT	12
10 Month Cohort
Condition	Genotype	N
Exercise	Pinkl−/−	14
Sham Exercise	Pinkl−/−	8
Sham Exercise	WT	8

2.2: Tongue Exercise

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 0.2g of force with their tongue unto an aluminum force disk (18mm) (Sensotec load cell, 0–250g 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^60–63. 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.

2.2.1: Tongue Force and Timing Behaviors Testing & Analysis

The MVTF tongue force data point (mN) was calculated from a three-day average of the highest press force during tongue exercise sessions (see above). We also assessed changes 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^57,64.

2.3: Vocal Exercise

On each exercise day, vocal exercise was completed following tongue exercise. A well-established, reliable social mating paradigm was used to elicit male vocalizations^50,54,65. 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 minutes (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-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 administered⁵⁴ 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 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 15cm 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.

2.3.1: 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 (kilohertz-kHz) of the call. This methodology has been validated in prior research^50,66–68.

2.4: 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.

2.4.1: 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 minutes. 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 methodology⁵⁷.

2.4.2: Immunohistochemistry

Brainstem tissue reserved for 5-HT staining was processed as previously described^50,69. In brief, slices underwent a series of brief washes in 0.1M PBS, were incubated in 0.5% H2O2 and 0.1M PBS solution, and were washed in 0.1M PBS and PBS-T. 20% normal goat serum with PBS-T was used to block tissue for 1 hour. 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 hours. After incubation, tissue was washed in PBS-T, then AB solution (VECTASTAIN^® Elite ABC-HRP Kit, Peroxidase (Goat IgG)) was applied for 1 hour to tag secondary antibody. Following washes in 0.1M 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).

2.5: 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 ImageJ⁷⁰(U.S. National Institutes of Health, Bethesda, MD).

To conduct the analysis, RBG color images were converted to grayscale (8-bit), and a 350 × 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 × 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:

$$\mathit{\text{ROD}}=\left(\mathit{\text{average OD from ROI of each hemisphere}}\right)-\mathit{\text{OD from region devoid of stain}}$$

The resulting number was then averaged for each rat, per region of interest, across sections.

2.6: 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.

3: Results

3.1: Six Month Vocalization and Tongue Behavior Results

3.1.1: Ultrasonic Vocalization

There was a significant main effect of condition on the average peak frequency of FM calls (F(2,33)=45.26, p<0.0001). In post-hoc testing, the average peak frequency of FM calls was higher for the WT sham cohort than for the Pink1−/− exercise and sham cohorts (estimated difference=8374, p<0.0001, and estimated difference=8.745, p<0.0001, respectively) (Figure 1A, Supplemental Table 1).

Figure 1:

USV changes with exercise at 6 months. A: Average peak frequency of FM calls at the 6 month time point. WT sham rats produced higher average peak frequency than both Pink1−/− exercise and sham groups. B: Intensity of FM calls at 6 months. There were no significant differences across groups or time points on measures of intensity. C: Proportion of Complex calls at 6 months. Pink1−/− rats that underwent vocal and tongue exercise produced a greater proportion of complex calls than WT rats in the sham condition. Data points represent group mean at each time point. Error bars represent +/−SEM. Hz: Hertz; dB: decibels.

There were no interaction effects between condition and baseline to final time points(F(2,33)=1.003, p=0.378), nor were there main effects of either time point(F(1,33)=0.105, p=0.747) or condition on average intensity of FM calls (F(2,33)=3.252, p=0.051) (Figure 1B, Supplemental Table 1).

There was a significant main effect of condition on the proportion of complex calls (F(2,33)=5.25, p=0.011). In post-hoc testing, the Pink1−/− exercise group produced a greater proportion of complex calls than the WT sham group (estimated difference=0.123, p=0.015). There was also a significant main effect of time point on the proportion of complex calls (F(1,33)=8.73, p=0.006). Across cohorts, rats produced a greater proportion of complex calls at baseline than at the final time point (estimated difference=0.067, p=0.006) (Figure 1C, Supplemental Table 1).

3.1.2: Tongue Force and Coefficient of Variation

There was a significant interaction between condition and time point on maximum press force (F(2,42)=5.6, p=0.007). In post-hoc testing, there was a significant increase in maximum press force following exercise for the Pink1−/− exercise cohort (estimated difference=50.72mN, p=0.003). The Pink1−/− exercise cohort also produced greater maximum press force at the final time point than the Pink1−/− sham, and the WT sham cohorts (estimated difference=42.86mN, p=0.0446, and estimated difference=81.62mN, p=0.0001, respectively). In addition, the Pink1−/− sham cohort produced greater maximum press force at the final time point than the WT sham cohort (estimated difference=38.76mN, p=0.0446). Other comparisons were non-significant or not experimentally relevant (i.e., Pink1−/− exercise force at the final time point versus WT sham force at baseline) (Figure 2, Supplemental Table 3).

Figure 2:

A: Maximum lingual press force at 6 months. Pink1−/− rats that underwent exercise produced greater maximum press force at the final time point than at baseline. B: Coefficient of variation in press force at 6 months. Pink1−/− rats in both exercise and sham cohorts had greater coefficient of variation in press force than the WT cohort, and coefficient of variation was greater at the final time point than at baseline for all cohorts. Data points represent group mean at each time point Error bars represent +/−SEM. mN: millinewtons

There was a significant main effect of treatment on coefficient of variation of press force at 6 months (F(2,42=17.59, p<0.0001). In post-hoc testing, the Pink1−/− exercise cohort and the Pink1−/− sham cohort both had greater coefficients of variation than the WT sham cohort (estimated difference=0.59, p=0.0006, and estimated difference=0.78, p<0.0001, respectively). There was not a significant difference in coefficient of variation between the Pink1−/− exercise and sham cohorts (estimated difference=0.19, p=0.22).

There was also a significant main effect of time point on coefficient of variation of press force at 6 months (F(1,42)=9.87, p=0.003). In post-hoc testing, the coefficient of variation was greater at the final time point than at baseline (estimated difference=0.24, p=0.003) (Figure 2, Supplemental Table 3).

Experimentally relevant post-hoc comparisons assessing press force across 30 second time intervals are summarized below and are visualized in Figure 3. All post-hoc comparisons are adjusted for multiple comparisons using the Holm-Sidak method.

Figure 3:

Changes to tongue press force over the course of a testing session A. baseline and B. final time points at 6 months. Data points represent group mean at each time point Error bars represent +/−SEM. mN: millinewtons.

There was a significant main effect of condition on press force at 30 seconds (F(2,42)=6.521, p=0.003. In post-hoc testing there Pink1−/− rats in the exercise cohort produced significantly greater pressure than the WT sham cohort (estimated difference=5.29mN, p=0.004). There were no significant differences between the Pink1−/− exercise and sham cohorts, or between the Pink1−/− sham and WT sham cohorts.

At 60 seconds, there was a significant interaction between condition and time point on press force (F(2,39=6.05, p=0.005). In post-hoc testing, there was a significant increase in press force from baseline to the final time point for the Pink1−/− exercise cohort (estimated difference=11.01mN, p=0.012). Neither the Pink1−/− sham nor the WT sham cohorts produced significant differences in press force between the baseline and final time points. At the final time point, the Pink1−/− exercise and sham cohorts both had greater press force than the WT sham cohort (estimated difference=18.16mN, p=0.0003, and estimated difference=13.95mN, p=0.004, respectively). There was not a difference between the two Pink1−/− cohorts at the final time point.

At 90 seconds, there was a significant interaction between condition and time point on press force (F(2,38)=10.86, p=0.0002). In post-hoc testing, both the Pink1−/− exercise and sham cohorts demonstrated significant increases in press force at the final time point compared to baseline (estimated difference=24.42mN, p<0.0001 and estimated difference=10.62mN, p=0.03, respectively). The WT sham cohort did not produce significant differences in press force between baseline and final time points. At the final time point, the Pink1−/− exercise and sham cohorts both produced greater press force than the WT sham cohort (estimated difference=31.79mN, p<0.0001, and estimated difference=19.51, p=0.006, respectively). There was not a difference between the two Pink1−/− cohorts at the final time point.

At 120 seconds, there was a significant interaction between condition and time point on press force (F(2,32)=7.04, p=0.003). In post-hoc testing, the Pink1−/− exercise cohort demonstrated a significant increase in press force at the final time point compared to baseline (estimated difference=26.69mN, p=0.004). Both the Pink1−/− exercise and sham cohorts produced greater press force at the final time point than the WT sham cohort (estimated difference=38.36mN, p=0.0001, and estimated difference=26.91mN, p=0.008, respectively), but the two Pink1−/− cohorts did not differ from one another.

At 150 seconds, there was a significant interaction between condition and time point on press force (F(2,26)=11.66, p=0.0002). In post-hoc testing, both the Pink1−/− exercise and sham cohorts demonstrated significant increases in press force at the final compared to baseline time point (estimated difference=35.61mN, p=0.0001, and estimated difference=19.72mN, p=0.02, respectively). At the final time point, both the Pink1−/− exercise and sham cohorts produced greater press force than the WT cohort (estimated difference=44.43, p<0.0001, and estimated difference=23.32, p=0.01, respectively). The Pink1−/− exercise cohort also produced significantly greater press force than the Pink1−/− sham cohort at the final time point (estimated difference=21.1mN, p=0.04).

At 180 seconds, there was a significant interaction between condition and time point on press force (F2,19)=7.81, p=0.003). In post-hoc testing, the Pink1−/− exercise cohort produced significantly greater force at the final time point compared to baseline (estimated difference=50.7mN, p=0.0009). At the final time point, the Pink1−/− exercise cohort produced greater force than the WT cohort (estimated difference=47.39mN, p=0.0002). The Pink1−/− sham cohort did not produce significantly different press force than either the Pink1−/− exercise or the WT sham cohorts.

At 210 seconds, there was a significant interaction between condition and time point on press force (F(2,10)=14.58, p=0.001). In post-hoc testing, the Pink1−/− exercise cohort produced significantly greater force at the final time point compared to baseline (estimated difference=83.38mN, p=0.0006). At the final time point, the Pink1−/− exercise cohort produced significantly greater press force than both the Pink1−/− and the WT sham cohorts (estimated difference=57.88mN, p=0.0002, and estimated difference=78.28mN, p<0.0001, respectively). The WT sham and Pink1−/− sham cohorts did not differ from one another at the final time point.

At 240 seconds, there was a significant main effect of treatment (F(2,24)=6.15, p=0.007). In post-hoc testing, there the Pink1−/− exercise cohort produced greater press force than both the Pink1−/− sham and WT sham cohorts (estimated difference=22.26mN, p=0.012, and estimated difference=31.02mN, p=0.001, respectively). The Pink1−/− sham and WT sham cohorts did not differ significantly from one another. There was also a significant main effect of time point on press force (F(1,24)=9.68, p=0.005), but the 2 time points did not differ significantly in post-hoc testing (estimated difference=34.8mN, p=0.08).

At 270 and 300 seconds, the number of rats that produced measurable press force across conditions was insufficient for statistical analyses.

3.2: Ten Month Vocalization and Tongue Behavior Results

3.2.1: Ultrasonic Vocalization

There was a significant main effect of condition on the average peak frequency of FM calls (F(2,34)=7.63, p=0.002). In post-hoc testing, the average peak frequency of FM calls was higher for the WT sham cohort than for the Pink1−/− exercise and sham cohorts (estimated difference=3847, p=0.016, and estimated difference=5911, p=0.0014, respectively) (Figure 4A, Supplemental Table 2).

Figure 4:

USV changes with exercise at 10 months. A: Average peak frequency of FM calls at the 10 month time point. WT sham rats produced louder calls than both Pink1−/− exercise and sham groups. B: Intensity of FM calls at 10 months. There were no significant different differences across groups or time points on measures of intensity. C: Proportion of complex calls at 10 months. There were no differences in proportion of complex calls across groups or between time points. Data points represent group mean at each time point Error bars represent +/−SEM. Hz: Hertz; dB: decibels.

There were no interaction effects between condition and time point (F(2,34)=0.575, p=0.454), nor were there main effects of either time point (F(1,34)=0.003, p=0.96) or condition (F(2,34)=2.25, p=0.121) on average intensity of FM calls (Figure 4B, Supplemental Table 2).

There were no interaction effects between condition and time point (F(2,34)=0.362, p=0.55), nor were there main effects of either time point (F(1,34)=0.242, p=0.626) or condition (F(2,34)=3.012, p=0.063) on proportion of complex calls (Figure 4C, Supplemental Table 2).

3.2.2: Tongue Force and Coefficient of Variation

There was a significant interaction between condition and time point (F(2,27)=26.77, p<0.0001). In post-hoc testing, there was a significant increase in maximum press force following exercise for the Pink1−/− exercise cohort (estimated difference=60.19mN, p<0.0001). At baseline, the Pink1−/− exercise and sham cohorts produced greater maximum press force than the WT sham cohort (estimated difference=71.98mN, p<0.0001, and estimated difference=56.8mN, p=0.0001, respectively). At the final time point, the Pink1−/− exercise cohort also produced greater maximum press force than the Pink1−/− sham and WT sham cohorts (estimated difference=75.36mN, p<0.0001, and estimated difference=121.73mN, p<0.0001, respectively). In addition, the Pink1−/− sham cohort produced greater maximum press force than the WT sham cohort at the final time point (estimated difference=62.47mN, p<0.0001). Other comparisons were non-significant or not experimentally relevant (i.e., Pink1−/− exercise force at the final time point versus WT sham force at baseline) (Figure 5, Supplemental Table 4).

Figure 5:

A: Maximum lingual press force at 10 months. Pink1−/− rats that underwent exercise produced greater maximum press force at the final time point than at baseline. B: coefficient of variation in press force at 10 months. Data points represent group mean at each time point Error bars represent +/−SEM. mN: millinewtons

There was a significant interaction between treatment and time point on coefficient of variation in tongue force at 10 months (F(2,27)=14.49, p<0.0001). In post-hoc testing, The Pink1−/− exercise cohort had a lower coefficient of variation than the Pink1−/− sham cohort at baseline (estimated difference=0.45, p=0.005). The Pink1−/− exercise cohort had a greater coefficient of variation than the Pink1−/− sham and WT sham cohorts at the final time point, but this did not reach statistical significance (estimated difference=0.35, p=0.06, and estimated difference=0.31, p=0.1, respectively). The Pink1−/− sham cohort, but not the WT sham cohort, demonstrated significant decreases in coefficient of variation of press force at the final time point compared to baseline (estimated difference=0.55, p=0.003, and estimated difference=0.4, p=0.06). Conversely, Pink1−/− exercise cohort demonstrated an increase in coefficient of variation in press force at the final time point compared to baseline, but this also did not reach statistical significance (estimated difference=0.24, p=0.15) (Figure 5, Supplemental Table 5).

Experimentally relevant post-hoc comparisons assessing press force across 30 second time intervals are summarized below and visualized in Figure 6. All post-hoc comparisons are adjusted for multiple comparisons using the Holm-Sidak method.

Figure 6:

Changes to tongue press force over the course of a testing session A. baseline and B. final time points at 10 months. Data points represent group mean at each time point Error bars represent +/−SEM. mN: millinewtons.

At 30 seconds, there was a significant main effect of condition on press force (F(2,27)=9.08, p=0.001). In post-hoc testing both the Pink1−/− exercise and sham cohorts produced greater press force than the WT sham cohort (estimated difference=5.28mN, p=0.0007, and estimated difference=3.6mN, p=0.03, respectively).

At 60 seconds, there was a significant interaction between condition and time point on press force (F(2,27)=4.26, p=0.03). In post-hoc testing, the Pink1−/− exercise cohort produced significantly greater force at the final time point compared to baseline (estimated difference=17.34mN, p<0.0001).

At 90 seconds, there was a significant interaction between condition and time point on press force (F(2,27)=3.58, p=0.04). In post-hoc testing, both the Pink1−/− exercise and sham cohorts produced greater press force than the WT sham cohort at baseline (estimated difference=21.97mN, p<0.0001, and estimated difference=15.51mN, p=0.006, respectively), and at the final time point (estimated difference=13.38mN, p=0.008, and estimated difference=16.44mN, p=0.004, respectively). The two Pink1−/− cohorts did not differ significantly from one another at either time point, and neither demonstrated a significant change in press force between baseline and final time points.

At 120 seconds, there was a significant main effect of condition on press force (F(2,26)=15.07, p<0.0001). In post-hoc testing, both the Pink1−/− exercise and sham cohorts produced greater press force than the WT cohort (estimated difference= 21.6mN, p<0.0001, and estimated difference=19.39mN, p=0.0005, respectively). The two Pink1−/− cohorts did not differ from one another. There was also a significant main effect of time point on press force (F(11,25)=4.7, p=0.04), however, there were no significant differences in press force between time points in post-hoc testing (estimated difference=7.04mN, p=0.07).

At 150 seconds, there was a significant main effect of condition on press force (F(2,24)=7.91, p=0.002). In post-hoc testing, both the Pink1−/− exercise and sham cohorts produced greater press force than the WT sham cohort (estimated difference=24.98mN, p=0.005, and estimated difference=30.11mN, p=0.005, respectively). There was also a significant main effect of time point on press force (F(1,20)=23.27, p=0.0001). In post-hoc testing, there was a significant increase in press force at the final time point (estimated difference=18.6mN, p=0.0009).

At 180 seconds, there was a significant interaction between condition and time point on press force (F(2,19)=4.45, p=0.03). In post-hoc testing, the Pink1−/− exercise cohort produced significantly greater press force at then final time point than at baseline (estimated difference=33.4mN, p=0.0001). At the final time point, the Pink1−/− exercise cohort also produced greater force than the WT sham cohort (estimated difference=38.97mN, p=0.006). There was not a significant difference between the Pink1−/− exercise and sham cohorts, or between the Pink1−/− sham and WT sham cohorts at the final time point.

At 210 seconds, there was a significant main effect of condition on press force (F(2,18)=4.58, p=0.03). In post-hoc testing, the Pink1−/− exercise cohort produced greater press force than the WT sham cohort (estimated difference=34.74mN, p=0.02). There was also a significant main effect of time point on press force (F(1,16)=16.96, p=0.0008). In post-hoc testing, press force was significantly greater at the final time point compared to baseline (estimated difference=22.6mN, p=0.007).

At 240 seconds, there was a significant main effect of condition on press force (F(2,19)=4.98, p=0.02). In post-hoc testing, both the Pink1−/− exercise and sham cohorts produced greater press force than the WT sham cohort (estimated difference=36.45mN, p=0.04, and estimated difference=39.11mN, p=0.04, respectively). There was also a significant main effect of time point on press force (F(1,14)=10.63, p=0.006). In post-hoc testing, however, there was not a significant difference in press force between time points (estimated difference=12.2mN, p=0.25).

At 270 seconds, there was a significant main effect of time point on press force (F(1,9)=8.27, p=0.02). In post hoc testing, however, there was not a significant difference in press force between time points (estimated difference=31.4mN, p=0.28).

Statistical analysis was not completed at 300 seconds because neither the WT sham nor the Pink1−/− sham cohorts produced measurable press force at baseline.

3.3: Six-Month Immunohistochemistry

Figure 7 demonstrates appropriate staining of brainstem 5HT in a wild-type sham rat. There was a significant effect of group on relative optical density in the 12N (Kruskal-Wallis χ²=8.3244, p=0.01557). In Bonferroni-corrected post-hoc testing, relative optical density was significantly greater for Pink1−/− rats without exercise than for WT control rats (p=0.036). Pink1−/− rats with and without exercise were not significantly different from one another, and WT control rats were not significantly different from Pink1−/− rats with exercise (p=0.20 and p=0.282, respectively) (Figure 8a).

Figure 7:

Photomicrograph of 5HT staining in the brainstem of a rat in the WT Sham condition at 6 months, demonstrating expected distribution of staining. 12N Box is placed in the hypoglossal nucleus. ROb box is placed immediately lateral to the Raphe Obscurus. Bregma=−13.32mm. Scale bar: 500um.

Figure 8:

Relative optical density of 5HT A) 12N at 6 months, B) Raphe Obscurus at 6 months, C) 12N at 10 months, and D) Raphe Obscurus at 10 months. Relative optical density: relative optical density; 12N: hypoglossal nucleus; Raphe Obs: Raphe Obscurus. Box borders represent the 25^th and 75^th percentiles; horizontal line in center of boxes represents 50^th percentile. Whiskers represent 1.5x the interquartile range + the 75^th percentile or – the 25^th percentile. Dots represent statistical outliers. *=p<0.05; **=p<0.01; ***=p<0.001

There were no significant differences in relative optical density in the Raphe Obscurus at the 6 month time point among groups (F(2,4.4)=1.1281, p=0.401) (Figure 8b).

3.4: Ten-month Immunohistochemistry

There was a significant effect of condition on relative optical density in the 12N at the 10 month time point, with a large effect size (F(2,22)=13.259, p=0.000167; η2=0.547). In post-hoc testing, relative optical density was significantly greater for the Pink1−/− sham exercise group than for the Pink1−/− exercise group (mean difference=0.0826, p=0.0002), and for the WT sham exercise group than for the Pink1−/− exercise group (mean difference =0.0652, p=0.00562). There was not a significant difference between the WT sham exercise and Pink1−/− sham exercise groups (Figure 8c).

There was a significant effect of condition on relative optical density in the Raphe Obscurus at the 10-month time point (Kruskal-Wallis χ²=11.62, p=0.002997). In post-hoc testing with Bonferroni correction, relative optical density was significantly lower for the Pink1−/− exercise group than for the Pink1−/− Sham (adjusted p=0.0048) and the WT Sham (adjusted p=0.0351) groups. The Pink1−/− sham and WT groups were not significantly different from one another (Figure 8d).

4.0: Discussion

This study demonstrates the effects of vocal and tongue exercise on ultrasonic vocalization and tongue force, as well as on serotonergic brainstem nuclei in the Pink1−/− rat model of PD. Importantly, this is observed at stages of disease progression that reflect the time when patients with PD often begin exercises targeted at vocal and swallowing deficits. In contrast to tongue and vocal exercise at earlier time points (4 months of age)⁵⁷, and in accordance with effects of exercise in patients with PD, we observed less robust effects with a relatively large degree of variability in outcomes later in the disease process^37,38,71,72.

Vocalization

In replication of previous work, we observed that intensity and average peak frequency of FM calls were greater for the WT sham group than for either of the Pink1−/− groups^28,50,51, suggesting that tandem vocal and tongue exercise at the 6 and 10 month time points did not ameliorate these vocal deficits in this model of PD. In contrast, vocal complexity was significantly greater at the 6-month time point for Pink1−/− rats that underwent exercise than for WT rats that underwent sham exercise. The significance of this in our model of vocal deficits in PD is unclear, as the proportion of complex calls was not different between the WT sham and Pink1−/− sham groups. However, greater call complexity may have ethological validity, as greater call complexity is associated with increased approach behavior in female conspecifics^68,73. The observed increase in call complexity could also be analogous to therapy in humans that targets increases in intelligibility through compensatory strategies such as clearer, more-precise articulation, without necessarily modulating underlying vocal deficit (reduced vocal intensity) or underlying neurobiological pathophysiology. Future studies would benefit from monitoring the behavioral effects of increased proportion of complex calls on approach behavior in mating conspecifics, as these increases may mitigate the decreased approach behavior that is associated with reduced average peak frequency⁷³.

Of note, the greater call complexity in the Pink1−/− exercise cohort at 6 months was not observed at 10 months. This, too, reflects the human response to behavioral intervention for treatment of vocal deficits in PD: efficacy is reduced at later time points in disease progression⁷¹. A potential explanation for this finding is that the ability to alter motor behavior (“motor learning”) may be reduced with disease progression in PD⁷⁴. If this is the case, early implementation of exercise programs would be more likely to have a positive influence on vocalization outcomes than exercise initiated later in disease progression. Future work should assess the duration of the benefit of vocal exercise initiated at the 6-month time point and earlier. In addition, longer duration of exercise or continuous exercise may lead to sustained benefit over time. Indeed, this is the case in treatment of vocal deficits in patients with PD^75,76. Assessment of the effects of continuous exercise would then help to deepen our understanding of this model of PD, and serve as a platform for studying ways to maintain the benefits of an initial episode of exercise without the burden of continuous exercise.

Tongue Press Force

Reflecting improvements in tongue strength following tongue exercise in patients with PD^40–42, we observed that maximum press force increased with exercise at both the 6-month and 10-month time points in the Pink1−/− rat model. This finding is promising in that it demonstrates that change in tongue force production is possible in later stages of disease progression. This is also clinically significant because increases in tongue strength with exercise have been correlated with improved swallow function in patients with PD^40–42. Thus, a potential clinical implication could be greater focus on tongue exercise in later stages of disease progression in humans, with a goal of reducing swallow impairment, and the associated increases in aspiration pneumonia and mortality. The functional impact of increased force in the Pink1−/− rat model of PD has yet to be determined, and future work would benefit from assessing possible relationships between lingual force production and functional swallowing measurements, such as swallowing kinematics measured during videofluoroscopy using the computational analysis of swallowing mechanics-rodent (CASM-R)⁷⁷.

Coefficient of variation in press force remained stable between the baseline and final time points in the 6-month cohort. In the 10-month cohort, however, we observed decreases in coefficient of variation in press force from baseline to the final time point for the WT and Pink1−/− sham cohorts, and an increase in coefficient of variation for the Pink1−/− exercise cohort. It is possible that this represents a simple regression toward the mean, or that increases in the coefficient of variation for the exercise cohort are a consequence of the greater range of force generation that becomes possible with progressive exercise. An additional potential explanation for this finding is that the increase in coefficient of variation in the exercise cohort represents a compensatory strategy for avoiding fatigue with the increasing force that was mandated as part of the exercise paradigm. If this is the case, it could reflect compensatory strategies used in the treatment of swallowing deficits, which includes slowing the rate of oral intake and resting as necessary to avoid fatigue⁷⁸. Larger sample sizes that can account for changes in time over the course of a testing episode will help to further clarify this observation.

There were also differences in press force across conditions that were more obvious later in individual testing episodes following exercise or sham exercise, particularly from 120 to 240 seconds, with the Pink1−/− exercise cohort demonstrating the greatest press force during these portions of the testing episode. This was true for both the 6-month and 10-month cohorts, and implies improved endurance as well as improved capacity for force generation. This is an important finding for clinical translation, as it implies that tongue exercise could improve swallow function over the duration of a meal, mitigating the increased risks of prandial aspiration and malnutrition characteristic of PD.

Exercise was associated with reduced 5HT in the 12N at the 10-month time point. At the 6-month time point, the relative optical density for 5HT was very similar between the WT and Pink1−/− Exercise groups. At the latter time point, however, the Pink1−/− exercise rats had lower 5HT than both the WT and Pink1−/− sham groups. While the relationship between exercise and 12N 5HT immunoreactivity is unclear, it demonstrates that neurochemical changes correspond to changes in tongue force in a rat model of cranial sensorimotor deficits in PD. Interestingly, our findings partially contrast with previous work that has demonstrated changes in 5HT in the 12N with a similar tongue exercise protocol in WT rats³⁴. While we observed decreases in 5HT in the 12N at 10 months of age in Pink1−/− rats that underwent tongue exercise compared to sham exercise, Behan and colleagues (2012) observed increases in 5HT in the 12N compared to sham exercise. In vivo experimentation would help to clarify mechanistic relationships between exercise and 12N 5HT changes, and application of exercise to a WT group would help to determine whether this type of neural and physiological plasticity is equally present across genotypes. It is also important to note that in the Behan et al 2012 study tongue exercise performed in older rats (24 months and 33 months) resulted in no change in 5HT in the 12N compared to sham exercise. This suggests the potential for interactions among age, disease progression and exercise that should be explored in future work. An additional potential explanation for differences observed in the current study is that tongue exercise was not performed in isolation, but in tandem with vocal exercise, which could have had a moderating influence on brainstem 5HT changes. Finally, in the current study, 5HT immunoreactivity was averaged across all the entire 12N. Analysis of changes to brainstem serotonergic systems in different subdivisions of the 12N should be considered in future work using larger sample sizes. Similar to the 12N, 5HT in the raphe obscurus was reduced with exercise, although the differences among groups did not reach statistical significance at the 6-month time point. Collectively, these neurochemical findings demonstrate that portions of the serotonergic system relevant to lingual movement are altered following exercise. Future pharmacologic investigations assessing the influence of serotonin modulators on lingual function in this model of PD may open new avenues of investigation for optimizing treatment of swallowing deficits in PD.

A relative strength of this study is the behavioral variability observed across all rats, regardless of condition. In mirroring the behavioral variability present in patients who undergo voice and swallowing exercises, we can see that, as in patient populations, most, but not all individuals improve with exercise. Unearthing the cause for this variable improvement, whether behavioral or neurobiological in nature, is critical for improving voice and swallowing outcomes in patients with PD. Future work using this model of voice and swallowing deficits in PD should account for and intentionally manipulate both of these factors through pharmacological and behavioral interventions.

This study is limited by its exclusion of female rats. The purpose of this exclusion was to increase experimental control in this early investigation of cranial sensorimotor exercise in a genetic model of PD. An immediate future direction will be to include female rats, controlling for stage of estrous cycle, not only at testing time points, but at each targeted exercise session, as stage of estrous cycle will likely influence outcomes of interest. Previous work assessing only female Pink1−/− rats has demonstrated important gender-based differences in this model of cranial sensorimotor deficits in PD, including lack of progression of vocalization deficits⁵¹. Inclusion of sex (and estrous cycle) as covariates in a single study, however, is likely to lead to greater insights into the disease process, responsiveness to exercise, and the potential neuroprotective properties of sex hormones^79–81.

5: Conclusion

Vocal and tongue exercises alter vocalization and tongue function at 6 and 10 months of age in the Pink1−/− rat model of vocal and swallowing deficits in PD, and completion of these exercises is also associated with changes to serotonergic immunoreactivity in the brainstem. Changes to vocalization with exercise are less robust at these later time points compared to exercise completed in prodromal stages of disease progression References

Highlights.

• Vocal and lingual exercise have a reduced impact on vocalization at 6 and 10 months of age in the Pink1−/− rat model of PD compared to 4 months of age.

• Exercise increases tongue force generation at both 6- and 10-month time points.

• Exercise is associated with decreased 5HT brainstem immunoreactivity.

Abbreviations:

PD

Parkinson disease

Pink1−/−

PTEN-induced putative kinase 1 gene knockout

USV

ultrasonic vocalization

WT

wild type

5HT

serotonin

MVTF

maximum voluntary tongue force