Prodromal Parkinson disease signs are predicted by a whole-blood inflammatory transcriptional signature in young Pink1-/- rats.

BACKGROUND: Parkinson disease (PD) is the fastest growing neurodegenerative disease. The molecular pathology of PD in the prodromal phase is poorly understood; as such, there are no specific prognostic or diagnostic tests. A validated Pink1 genetic knockout rat was used to model early-onset and progressive PD. Male Pink1-/- rats exhibit progressive declines in ultrasonic vocalizations as well as hindlimb and forelimb motor deficits by mid-to-late adulthood. Previous RNA-sequencing work identified upregulation of genes involved in disease pathways and inflammation within the brainstem and vocal fold muscle. The purpose of this study was to identify gene pathways within the whole blood of young Pink1-/- rats (3 months of age) and to link gene expression to early acoustical changes. To accomplish this, limb motor testing (open field and cylinder tests) and ultrasonic vocalization data were collected, immediately followed by the collection of whole blood and RNA extraction. Illumina® Total RNA-Seq TruSeq platform was used to profile differential expression of genes. Statistically significant genes were identified and Weighted Gene Co-expression Network Analysis was used to construct co-expression networks and modules from the whole blood gene expression dataset as well as the open field, cylinder, and USV acoustical dataset. ENRICHR was used to identify the top up-regulated biological pathways. RESULTS: The data suggest that inflammation and interferon signaling upregulation in the whole blood is present during early PD. We also identified genes involved in the dysregulation of ribosomal protein and RNA processing gene expression as well as prion protein gene expression. CONCLUSIONS: These data identified several potential blood biomarkers and pathways that may be linked to anxiety and vocalization acoustic parameters and are key candidates for future drug-repurposing work and comparison to human datasets.

Pink1-/- Rats Demonstrate Swallowing and Gastrointestinal Dysfunction in a Model of Prodromal Parkinson Disease.

Early motor and non-motor signs of Parkinson disease (PD) include dysphagia, gastrointestinal dysmotility, and constipation. However, because these often manifest prior to formal diagnosis, the study of PD-related swallow and GI dysfunction in early stages is difficult. To overcome this limitation, we used the Pink1-/- rat, a well-established early-onset genetic rat model of PD to assay swallowing and GI motility deficits. Thirty male rats were tested at 4 months (Pink1-/- = 15, wildtype (WT) control = 15) and 6 months (Pink1-/- = 7, WT = 6) of age; analogous to early-stage PD in humans. Videofluoroscopy of rats ingesting a peanut-butter-barium mixture was used to measure mastication rate and oropharyngeal and pharyngoesophageal bolus speeds. Abnormal swallowing behaviors were also quantified. A second experiment tracked barium contents through the stomach, small intestine, caecum, and colon at hours 0-6 post-barium gavage. Number and weight of fecal emissions over 24 h were also collected. Compared to WTs, Pink1-/- rats showed slower mastication rates, slower pharyngoesophageal bolus speeds, and more abnormal swallowing behaviors. Pink1-/- rats demonstrated significantly delayed motility through the caecum and colon. Pink1-/- rats also had significantly lower fecal pellet count and higher fecal pellet weight after 24 h at 6 months of age. Results demonstrate that swallowing dysfunction occurs early in Pink1-/- rats. Delayed transit to the colon and constipation-like signs are also evident in this model. The presence of these early swallowing and GI deficits in Pink1-/- rats are analogous to those observed in human PD.

Gene expression within the periaqueductal gray is linked to vocal behavior and early-onset parkinsonism in Pink1 knockout rats.

BACKGROUND: Parkinson's disease (PD) is a degenerative disease with early-stage pathology hypothesized to manifest in brainstem regions. Vocal deficits, including soft, monotone speech, result in significant clinical and quality of life issues and are present in 90% of PD patients; yet the underlying pathology mediating these significant voice deficits is unknown. The Pink1-/- rat is a valid model of early-onset PD that presents with analogous vocal communication deficits. Previous work shows abnormal α-synuclein protein aggregation in the periaqueductal gray (PAG), a brain region critical and necessary to the modulation of mammalian vocal behavior. In this study, we used high-throughput RNA sequencing to examine gene expression within the PAG of both male and female Pink1-/- rats as compared to age-matched wildtype controls. We used a bioinformatic approach to (1) test the hypothesis that loss of Pink1 in the PAG will influence the differential expression of genes that interact with Pink1, (2) highlight other key genes that relate to this type of Mendelian PD, and (3) catalog molecular targets that may be important for the production of rat vocalizations. RESULTS: Knockout of the Pink1 gene resulted in differentially expressed genes for both male and female rats that also mapped to human PD datasets. Pathway analysis highlighted several significant metabolic pathways. Weighted gene co-expression network analysis (WGCNA) was used to identify gene nodes and their interactions in (A) males, (B) females, and (C) combined-sexes datasets. For each analysis, within the module containing the Pink1 gene, Pink1 itself was the central node with the highest number of interactions with other genes including solute carriers, glutamate metabotropic receptors, and genes associated with protein localization. Strong connections between Pink1 and Krt2 and Hfe were found in both males and female datasets. In females a number of modules were significantly correlated with vocalization traits. CONCLUSIONS: Overall, this work supports the premise that gene expression changes in the PAG may contribute to the vocal deficits observed in this PD rat model. Additionally, this dataset identifies genes that represent new therapeutic targets for PD voice disorders.

Pink1 -/- Rats Show Early-Onset Swallowing Deficits and Correlative Brainstem Pathology.

Parkinson disease (PD) compromises oropharyngeal swallowing, which negatively affects quality of life and contributes to aspiration pneumonia. Dysphagia often begins early in the disease process, and does not improve with standard therapies. As a result, swallowing deficits are undertreated in the PD population. The Pink1 -/- rat is used to model PD, and demonstrates widespread brainstem neuropathology in combination with early-onset sensorimotor dysfunction; however, to date, swallowing behaviors have not been evaluated. To test the hypothesis that Pink1 -/- rats demonstrate early-onset differences in swallowing, we analyzed within-subject oropharyngeal swallowing using videofluoroscopy. Pink1 -/- and wildtype (WT) controls at 4 (Pink1 -/- n = 16, WT = 16) and 8 (Pink1 -/- n = 12, WT = 12) months of age were tested. The average and maximum bolus size was significantly increased in Pink1 -/- rats at both 4 and 8 months. Bolus average velocity was increased at 8 months for all animals; yet, Pink1 -/- animals had significantly increased velocities compared to WT at 8 months. The data show a significant reduction in mastication rate for Pink1 -/- rats at 8 months suggesting the onset of oromotor dysfunction begins at this time point. Relationships among swallowing variables and neuropathological findings, such as increased alpha-synuclein protein in the nucleus ambiguus and reductions in noradrenergic cells in the locus coeruleus in the Pink1 -/- rats, were determined. The presence of early oropharyngeal swallowing deficits and relationships to brainstem pathology in Pink1-/- rat models of PD indicate that this may be a useful model of early swallowing deficits and their mechanisms. These findings suggest clinical implications for early detection and management of dysphagia in PD.

Evidence for early and progressive ultrasonic vocalization and oromotor deficits in a PINK1 gene knockout rat model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease that leads to a wide range of motor and nonmotor deficits. Specifically, voice and swallow deficits manifest early, are devastating to quality of life, and are difficult to treat with standard medical therapies. The pathological hallmarks of PD include accumulation of the presynaptic protein α-synuclein (αSyn) as well as degeneration of substantia nigra dopaminergic neurons. However, there is no clear understanding of how or when this pathology contributes to voice and swallow dysfunction in PD. The present study evaluates the effect of loss of function of the phosphatase and tensin homolog-induced putative kinase 1 gene in rats (PINK1(-/-) ), a model of autosomal recessive PD in humans, on vocalization, oromotor and limb function, and neurodegenerative pathologies. Behavioral measures include ultrasonic vocalizations, tongue force, biting, and gross motor performance that are assayed at 2, 4, 6, and 8 months of age. Aggregated αSyn and tyrosine hydroxylase immunoreactivity (TH-ir) were measured at 8 months. We show that, compared with wild-type controls, PINK1(-/-) rats develop (1) early and progressive vocalization and oromotor deficits, (2) reduced TH-ir in the locus coeruleus that correlates with vocal loudness and tongue force, and (3) αSyn neuropathology in brain regions important for cranial sensorimotor control. This novel approach of characterizing a PINK1(-/-) genetic model of PD provides the foundational work required to define behavioral biomarkers for the development of disease-modifying therapeutics for PD patients.

Inverted-U shaped effects of D1 dopamine receptor stimulation in the medial preoptic nucleus on sexually motivated song in male European starlings.

Past studies in songbirds have highlighted a central role for the medial preoptic nucleus (mPOA) in context-appropriate vocal communication. During the breeding season, male songbirds sing primarily to attract females (sexually motivated song) and to repel competitors (agonistically motivated song). Past data have linked dopamine and D1 dopamine receptors in the mPOA to sexually motivated but not agonistically motivated song; however, direct effects of dopamine receptor manipulations in the mPOA on song have not been experimentally tested. Here, we tested the hypothesis that D1 receptor stimulation in the mPOA selectively influences sexually motivated male song, and the possibility that the effects of D1 receptor agonism differ at low and high doses. In a first study, breeding-condition male European starlings received infusions of saline or a single dose of the D1 receptor agonist SKF 38393 on separate test days into the mPOA or hypothalamic control areas. Stimulation of D1 receptors in the mPOA triggered sexually motivated but not agonistically motivated song. A second study showed inverted-U shaped dose-response effects of the agonist, such that low levels of sexually motivated song were observed at low and high levels of D1 receptor activation. A third study showed that the effects of the D1 receptor agonist were blocked by the D1 receptor antagonist SCH 23390. These findings suggest that an optimal level of D1 receptor stimulation in the mPOA is needed to facilitate sexually motivated vocal production. The results support a central, context-specific role for the mPOA in vocal communication, and more broadly demonstrate a complex, modulatory influence of D1 receptors in the mPOA on sexually motivated behavior.

Large-scale gene expression changes in APP/PSEN1 and GFAP mutation models exhibit high congruence with Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with both genetic and non-genetic causes. Animal research models are available for a multitude of diseases and conditions affecting the central nervous system (CNS), and large-scale CNS gene expression data exist for many of these. Although there are several models specifically for AD, each recapitulates different aspects of the human disease. In this study we evaluate over 500 animal models to identify those with CNS gene expression patterns matching human AD datasets. Approaches included a hypergeometric based scoring system that rewards congruent gene expression patterns but penalizes discordant gene expression patterns. The top two models identified were APP/PS1 transgenic mice expressing mutant APP and PSEN1, and mice carrying a GFAP mutation that is causative of Alexander disease, a primary disorder of astrocytes in the CNS. The APP/PS1 and GFAP models both matched over 500 genes moving in the same direction as in human AD, and both had elevated GFAP expression and were highly congruent with one another. Also scoring highly were the 5XFAD model (with five mutations in APP and PSEN1) and mice carrying CK-p25, APP, and MAPT mutations. Animals with the APOE3 and 4 mutations combined with traumatic brain injury ranked highly. Bulbectomized rats scored high, suggesting anosmia could be causative of AD-like gene expression. Other matching models included the SOD1G93A strain and knockouts for SNORD116 (Prader-Willi mutation), GRID2, INSM1, XBP1, and CSTB. Many top models demonstrated increased expression of GFAP, and results were similar across multiple human AD datasets. Heatmap and Uniform Manifold Approximation Plot results were consistent with hypergeometric ranking. Finally, some gene manipulation models, including for TYROBP and ATG7, were identified with reversed AD patterns, suggesting possible neuroprotective effects. This study provides insight for the pathobiology of AD and the potential utility of available animal models.

Tongue and laryngeal exercises improve tongue strength and vocal function outcomes in a Pink1-/- rat model of early Parkinson disease.

Parkinson disease (PD) causes voice and swallow dysfunction even in early stages of the disease. Treatment of this dysfunction is limited, and the neuropathology underlying this dysfunction is poorly defined. Targeted exercise provides the greatest benefit for offsetting voice and swallow dysfunction, and previous data suggest the hypoglossal nucleus and noradrenergic-locus coeruleus (LC) may be involved in its early pathology. To investigate relationships between targeted exercise and neuropathology of voice and swallow dysfunction, we implemented a combined exercise paradigm that included tongue force and vocalization exercises early in the Pink1-/- rat model. We tested the hypotheses that (1) tongue and vocal exercise improves tongue force and timing behaviors and vocalization outcomes, and (2) exercise increases optical density of serotonin (5-HT) in the hypoglossal nucleus, and tyrosine hydroxylase immunoreactive (Th-ir) cell counts in the LC. At two months of age Pink1-/- rats were randomized to exercise or non-exercise treatment. Age-matched wildtype (WT) control rats were assigned to non-exercise treatment. Tongue force and timing behaviors and ultrasonic vocalizations were measured at baseline (two months) and final (four months) timepoints. Optical density of 5-HT in the hypoglossal nucleus and TH-ir cell counts in the LC were obtained. Pink1-/- rats produced greater tongue forces, faster tongue contraction, and higher-intensity vocalization following exercise. There were no differences in LC TH-ir. The non-exercised Pink1-/- group had reduced density of 5-HT in the hypoglossal nucleus compared to the WT control group. The changes to tongue function and vocalization after targeted exercise suggests exercise intervention may be beneficial in early PD.

Early identification and treatment of communication and swallowing deficits in Parkinson disease.

Parkinson disease (PD) is a complex, progressive, neurodegenerative disorder that leads to a wide range of deficits including fine and gross sensorimotor impairment, autonomic dysfunction, mood disorders, and cognitive decline. Traditionally, the focus for diagnosis and treatment has been on sensorimotor impairment related to dopamine depletion. It is now widely recognized, however, that PD-related pathology affects multiple central nervous system neurotransmitters and pathways. Communication and swallowing functions can be impaired even in the early stages, significantly affecting health and quality of life. The purpose of this article is to review the literature on early intervention for communication and swallowing impairment in PD. Overarching themes were that (1) studies and interpretation of data from studies in early PD are limited; (2) best therapy practices have not been established, in part due to the heterogeneous nature of PD; and (3) as communication and swallowing problems are pervasive in PD, further treatment research is essential.

Effects of social isolation on 50-kHz ultrasonic vocalizations, affective state, cognition, and neurotransmitter concentrations in the ventral tegmental and locus coeruleus of adult rats.

Vocal communication, cognition, and affective state are key features of sustained health and wellness, and because vocalizations are often socially-motivated, social experience likely plays a role in these behaviors. The monoaminergic systems of the ventral tegmental area (VTA) and the locus coeruleus (LC) are associated with social and reward processing, vocalization production, and neurotransmitter changes in response to environmental stressors. The effect of social isolation on these complex behaviors and the underlying neural mechanisms is relatively unknown. To add to this body of literature, we randomized adult male Long-Evans rats to control (housed with a cagemate) or isolated (housed individually) conditions and assayed ultrasonic vocalizations, cognition (novel object recognition test), anxiety (elevated plus maze) and anhedonia (sucrose preference test) at 2, 4, 6, 8, and 10 months of age. At 10 months, VTA and LC samples were assayed for dopamine, norepinephrine, and serotonin using high performance liquid chromatography. We tested the hypotheses that isolation 1) diminishes vocalizations and cognition, 2) increases anxiety and depression, and 3) increases levels of dopamine, norepinephrine, and serotonin in the VTA and LC. Results showed isolation significantly reduced vocalization tonality (signal-to-noise ratio) and increased maximum frequency. There were no significant findings for cognition, anxiety, or anhedonia. Dopamine and serotonin and their respective metabolites were significantly increased in the VTA in isolated rats. These findings suggest chronic changes to social condition such as isolation affects vocalization production and levels of VTA neurotransmitters.

Prodromal Parkinson disease signs are predicted by a whole-blood inflammatory transcriptional signature in young Pink1-/- rats.

Background: Parkinson disease (PD) is the fastest growing neurodegenerative disease. The molecular pathology of PD in the prodromal phase is poorly understood; as such, there are no specific prognostic or diagnostic tests. A validated Pink1 genetic knockout rat was used to model early-onset and progressive PD. Male Pink1-/- rats exhibit progressive declines in ultrasonic vocalizations as well as hindlimb and forelimb motor deficits by mid-to-late adulthood. Previous RNA-sequencing work identified upregulation of genes involved in disease pathways and inflammation within the brainstem and vocal fold muscle. The purpose of this study was to identify gene pathways within the whole blood of young Pink1-/- rats (3 months of age) and to link gene expression to early acoustical changes. To accomplish this, limb motor testing (open field and cylinder tests) and ultrasonic vocalization data were collected, immediately followed by the collection of whole blood and RNA extraction. Illumina® Total RNA-Seq TruSeq platform was used to profile differential expression of genes. Statistically significant genes were identified and Weighted Gene Co-expression Network Analysis was used to construct co-expression networks and modules from the whole blood gene expression dataset as well as the open field, cylinder, and USV acoustical dataset. ENRICHR was used to identify the top up-regulated biological pathways. Results: The data suggest that inflammation and interferon signaling upregulation in the whole blood is present during early PD. We also identified genes involved in the dysregulation of ribosomal protein and RNA processing gene expression as well as prion protein gene expression. Conclusions: These data identified several potential blood biomarkers and pathways that may be linked to anxiety and vocalization acoustic parameters and are key candidates for future drug-repurposing work and comparison to human datasets.

Thyroarytenoid Oxidative Metabolism and Synaptic Signaling Dysregulation in the Female Pink1-/- Rat.

OBJECTIVES AND HYPOTHESIS: Vocal dysfunction, including hypophonia, in Parkinson disease (PD) manifests in the prodromal period and significantly impacts an individual's quality of life. Data from human studies suggest that pathology leading to vocal deficits may be structurally related to the larynx and its function. The Pink1-/- rat is a translational model used to study pathogenesis in the context of early-stage mitochondrial dysfunction. The primary objective of this work was to identify differentially expressed genes in the thyroarytenoid muscle and examine the dysregulated biological pathways in the female rat. METHODS: RNA sequencing was used to determine thyroarytenoid (TA) muscle gene expression in adult female Pink1-/- rats compared with controls. A bioinformatic approach and the ENRICHR gene analysis tool were used to compare the sequencing dataset with biological pathways and processes, disease relationships, and drug-repurposing compounds. Weighted Gene Co-expression Network Analysis was used to construct biological network modules. The data were compared with a previously published dataset in male rats. RESULTS: Significant upregulated pathways in female Pink1-/- rats included fatty acid oxidation and muscle contraction, synaptic transmission, and neuromuscular processes. Downregulated pathways included anterograde transsynaptic signaling, chemical synaptic transmission, and ion release. Several drug treatment options including cetuximab, fluoxetine, and resveratrol are hypothesized to reverse observed genetic dysregulation. CONCLUSIONS: Data presented here are useful for identifying biological pathways that may underlie the mechanisms of peripheral dysfunction including neuromuscular synaptic transmission to the TA muscle. These experimental biomarkers have the potential to be targeted as sites for improving the treatment for hypophonia in early-stage PD. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3412-3421, 2023.

Behavioral indices of breeding readiness in female European starlings correlate with immunolabeling for catecholamine markers in brain areas involved in sexual motivation.

In seasonally-breeding songbirds, lengthening photoperiod, increases in estradiol and exposure to male courtship facilitate breeding behavior in females in spring. However, there is extreme variability in the extent to which spring-condition females are attracted by male courtship or engage in nesting behavior. Here we explore possible links between catecholamines and individual differences in behaviors indicative of breeding readiness. Female European starlings were placed in conditions typical of the breeding season (spring-like) or the non-breeding season (fall-like). Although many females examined nesting locations, only a subset of spring-like females occupied nest sites. Labeling for dopamine-beta-hydroxylase (DBH; the enzyme involved in norepinephrine synthesis) in the ventromedial nucleus of the hypothalamus (VMH) was densest in females that acquired nest sites compared to spring-like females without nest sites or fall-like females. Within the group of spring-like females, nesting behaviors correlated positively with DBH labeling in VMH. Females with nest sites had the lowest density of DBH labeling in the ventral tegmental area, and labeling correlated negatively with spring-like female nesting behaviors. Labeling for tyrosine hydroxylase (TH; the rate limiting enzyme for catecholamine synthesis) in putative nucleus accumbens was lowest in spring-like females without nest sites, and labeling correlated positively with nesting behavior in spring-like females. TH labeling density in the medial preoptic nucleus was highest in fall-like females, but a trend was observed for a positive correlation between TH labeling and spring-like female nesting behaviors. These results link distinct patterns of catecholamine activity in brain regions implicated in sexual motivation to female breeding readiness.

Manipulation of vocal communication and anxiety through pharmacologic modulation of norepinephrine in the Pink1-/- rat model of Parkinson disease.

Vocal deficits and anxiety are common, co-occurring, and interacting signs of Parkinson Disease (PD) that have a devastating impact on quality of life. Both manifest early in the disease process. Unlike hallmark motor signs of PD, neither respond adequately to dopamine replacement therapies, suggesting that their disease-specific mechanisms are at least partially extra-dopaminergic. Because noradrenergic dysfunction is also a defining feature of PD, especially early in the disease progression, drug therapies targeting norepinephrine are being trialed for treatment of motor and non-motor impairments in PD. Research assessing the effects of noradrenergic manipulation on anxiety and vocal impairment in PD, however, is sparse. In this pre-clinical study, we quantified the influence of pharmacologic manipulation of norepinephrine on vocal impairment and anxiety in Pink1-/- rats, a translational model of PD that demonstrates both vocal deficits and anxiety. Ultrasonic vocalization acoustics, anxiety behavior, and limb motor activity were tested twice for each rat: after injection of saline and after one of three drugs. We hypothesized that norepinephrine reuptake inhibitors (atomoxetine and reboxetine) and a ß receptor antagonist (propranolol) would decrease vocal impairment and anxiety compared to saline, without affecting spontaneous motor activity. Our results demonstrated that atomoxetine and reboxetine decreased anxiety behavior. Atomoxetine also modulated ultrasonic vocalization acoustics, including an increase in vocal intensity, which is almost always reduced in animal models and patients with PD. Propranolol did not affect anxiety or vocalization. Drug condition did not influence spontaneous motor activity. These studies demonstrate relationships among vocal impairment, anxiety, and noradrenergic systems in the Pink1-/- rat model of PD.

Differences in dopamine and opioid receptor ratios in the nucleus accumbens relate to physical contact and undirected song in pair-bonded zebra finches.

Long-term social bonds are critical for survival and reproductive success in many species. Although courtship and pair-bond formation are relatively well studied, much less is known about the neural regulation of behaviors that occur after pair bonding that reinforce the bond and contribute to reproductive success. Dopamine and opioids in the nucleus accumbens (NAc) alter motivational state and reward by binding to receptor subtypes that engage distinct and opposing second messenger systems, and there is evidence that receptor ratios may influence social behavior. We used quantitative real-time PCR to explore relationships between messenger RNA ratios for dopamine D1 and D2 receptors (D1:D2) and mu and kappa opioid receptors (MOR:KOR) in NAc and behaviors implicated in reproductive investment and pair-bond maintenance in established male-female zebra finch pairs. In males, D1:D2 expression in NAc related negatively, whereas MOR:KOR related positively, to undirected song production. D1:D2 receptors also related positively to physical contact with a female. For females, D1:D2 expression was lower in females exposed to high compared to low rates of the partner's undirected song, and MOR:KOR expression in females related positively to undirected song exposure and allopreening. Analyses of single genes did not yield the same results. These findings suggest that the ratio of D1 to D2 and MOR to KOR receptor signaling in NAc causes differences in behavior or that behavior (or the partner's behavior) causes receptor ratio changes to modulate behaviors that maintain pair bonds and promote reproductive investment. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Predictors of prodromal Parkinson's disease in young adult Pink1-/- rats.

Parkinson's disease (PD) is a progressive, degenerative disease that affects nearly 10 million people worldwide. Hallmark limb motor signs and dopamine depletion have been well studied; however, few studies evaluating early stage, prodromal biology exist. Pink1-/- rats, a rodent model of PD mitochondrial dysfunction, exhibit early stage behavioral deficits, including vocal communication and anxiety, that progress during mid-to-late adulthood (6-12 months of age). Yet, the biological pathways and mechanisms that lead to prodromal dysfunction are not well understood. This study investigated the Pink1-/- rat in young adulthood (2 months of age). Mixed sex groups of Pink1-/- rats and wildtype (WT) controls were assayed for limb motor, anxiety, and vocal motor behaviors. A customized NanoString CodeSet, based on genetic work in later adulthood, was used to probe for the up regulation of genes involved in disease pathways and inflammation within the brainstem and vocal fold muscle. In summary, the data show sex- and genotype-differences in limb motor, anxiety, and vocal motor behaviors. Specifically, female Pink1-/- rats demonstrate less anxiety-like behavior compared to male Pink1-/- rats and female rats show increased locomotor activity compared to male rats. Pink1-/- rats also demonstrate prodromal ultrasonic vocalization dysfunction across all acoustic parameters and sex differences were present for intensity (loudness) and peak frequency. These data demonstrate a difference in phenotype in the Pink1-/- model. Tuba1c transcript level was identified as a key marker negatively correlated to ultrasonic vocalization at 2 months of age. Identifying genes, such as Tuba1c, may help determine early predictors of PD pathology in the Pink1-/- rat and serve as targets for future drug therapy studies.

Quantification of very late xerostomia in head and neck cancer patients after irradiation.

Objective: Radiation therapy (RT) for head and neck cancer (HNC) can result in severe xerostomia, or the subjective feeling of dry mouth. Characterizing xerostomia is critical to designing future clinical trials investigating how to improve HNC patients' quality of life (QoL). Few studies have investigated the very late (>5 years post-RT) effects of RT for HNC. We undertook preliminary studies quantifying very late xerostomia. Methods: Six adults who underwent RT for HNC at least 5 years prior and reported xerostomia were enrolled. Five healthy adults without a self-reported history of HNC or xerostomia were enrolled as controls. All participants completed three validated surveys to measure xerostomia-related QoL. Salivary production rates were measured and compositional analysis of the saliva and oral microbiome was completed. Results: The QoL survey scores for the HNC participants were significantly worse as compared to the control participants. The HNC participants produced less unstimulated saliva (p = .02) but not less stimulated saliva. The median salivary mucin significantly higher in HNC participants than in control participants (p = .02). There was no significant difference between the pH, amylase, or total protein. Microbiome analysis revealed alpha diversity to be significantly lower in the HNC participants. Conclusion: In the survivors of HNC who suffer from late toxicities, multiple means of measuring toxicity may be useful. We found that in patients with radiation-induced xerostomia over 5 years after therapy, not only were the QoL surveys significantly worse, as expected, but other measurements such as mucin and oral microbiome diversity were also significantly different. Level of evidence: 3.

Quantification of brainstem norepinephrine relative to vocal impairment and anxiety in the Pink1-/- rat model of Parkinson disease.

Vocal communication impairment and anxiety are co-occurring and interacting signs of Parkinson Disease (PD) that are common, poorly understood, and under-treated. Both vocal communication and anxiety are influenced by the noradrenergic system. In light of this shared neural substrate and considering that noradrenergic dysfunction is a defining characteristic of PD, tandem investigation of vocal impairment and anxiety in PD relative to noradrenergic mechanisms is likely to yield insights into the underlying disease-specific causes of these impairments. In order to address this gap in knowledge, we assessed vocal impairment and anxiety behavior relative to brainstem noradrenergic markers in a genetic rat model of early-onset PD (Pink1-/-) and wild type controls (WT). We hypothesized that 1) brainstem noradrenergic markers would be disrupted in Pink1-/-, and 2) brainstem noradrenergic markers would be associated with vocal acoustic changes and anxiety level. Rats underwent testing of ultrasonic vocalization and anxiety (elevated plus maze) at 4, 8, and 12 months of age. At 12 months, brainstem norepinephrine markers were quantified with immunohistochemistry. Results demonstrated that vocal impairment and anxiety were increased in Pink1-/- rats, and increased anxiety was associated with greater vocal deficit in this model of PD. Further, brainstem noradrenergic markers including TH and α1 adrenoreceptor immunoreactivity in the locus coeruleus, and ß1 adrenoreceptor immunoreactivity in vagal nuclei differed by genotype, and were associated with vocalization and anxiety behavior. These findings demonstrate statistically significant relationships among vocal impairment, anxiety, and brainstem norepinephrine in the Pink1-/- rat model of PD.

Thyroarytenoid Muscle Gene Expression in a Rat Model of Early-Onset Parkinson's Disease.

OBJECTIVES/HYPOTHESIS: Voice disorders in Parkinson's disease (PD) are early-onset, manifest in the preclinical stages of the disease, and negatively impact quality of life. The complete loss of function in the PTEN-induced kinase 1 gene (Pink1) causes a genetic form of early-onset, autosomal recessive PD. Modeled after the human inherited mutation, the Pink1-/- rat demonstrates significant cranial sensorimotor dysfunction including declines in ultrasonic vocalizations. However, the underlying genetics of the vocal fold thyroarytenoid (TA) muscle that may contribute to vocal deficits has not been studied. The aim of this study was to identify differentially expressed genes in the TA muscle of 8-month-old male Pink1-/- rats compared to wildtype controls. STUDY DESIGN: Animal experiment with control. METHODS: High throughput RNA sequencing was used to examine TA muscle gene expression in adult male Pink1-/- rats and wildtype controls. Weighted Gene Co-expression Network Analysis was used to construct co-expression modules to identify biological networks, including where Pink1 was a central node. The ENRICHR tool was used to compare this gene set to existing human gene databases. RESULTS: We identified 134 annotated differentially expressed genes (P < .05 cutoff) and observed enrichment in the following biological pathways: Parkinson's disease (Casp7, Pink1); Parkin-Ubiquitin proteasome degradation (Psmd12, Psmd7); MAPK signaling (Casp7, Ppm1b, Ppp3r1); and inflammatory TNF-α, Nf-κB Signaling (Casp7, Psmd12, Psmd7, Cdc34, Bcl7a, Peg3). CONCLUSIONS: Genes and pathways identified here may be useful for evaluating the specific mechanisms of peripheral dysfunction including within the laryngeal muscle and have potential to be used as experimental biomarkers for treatment development. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2874-E2879, 2021.

Rat Models of Vocal Deficits in Parkinson's Disease.

Parkinson's disease (PD) is a progressive, degenerative disorder that affects 10 million people worldwide. More than 90% of individuals with PD develop hypokinetic dysarthria, a motor speech disorder that impairs vocal communication and quality of life. Despite the prevalence of vocal deficits in this population, very little is known about the pathological mechanisms underlying this aspect of disease. As such, effective treatment options are limited. Rat models have provided unique insights into the disease-specific mechanisms of vocal deficits in PD. This review summarizes recent studies investigating vocal deficits in 6-hydroxydopamine (6-OHDA), alpha-synuclein overexpression, DJ1-/-, and Pink1-/- rat models of PD. Model-specific changes to rat ultrasonic vocalization (USV), and the effects of exercise and pharmacologic interventions on USV production in these models are discussed.