Tongue muscle plasticity following hypoglossal nerve stimulation in aged rats.

INTRODUCTION: Age-related decreases in tongue muscle mass and strength have been reported. It may be possible to prevent age-related tongue muscle changes using neuromuscular electrical stimulation (NMES). Our hypothesis was that alterations in muscle contractile properties and myosin heavy chain composition would be found after NMES. METHODS: Fifty-four young, middle-aged, and old 344/Brown Norway rats were included in this study. Twenty-four rats underwent bilateral electrical stimulation of the hypoglossal nerves for 8 weeks and were compared with control or sham rats. Muscle contractile properties and myosin heavy chain (MHC) in the genioglossus (GG), styloglossus (SG), and hyoglossus (HG) muscles were examined. RESULTS: Compared with unstimulated control rats, we found reduced muscle fatigue, increased contraction and half-decay times, and increased twitch and tetanic tension. Increased type I MHC was found, except for in GG in old and middle-aged rats. CONCLUSION: Transitions in tongue muscle contractile properties and phenotype were found after NMES.

High contrast, isotropic, and uniform 3D-imaging of centimeter-scale scattering samples using structured illumination light-sheet microscopy with axial sweeping.

Light-sheet fluorescent microscopy (LSFM) has, in recent years, allowed for rapid 3D-imaging of cleared biomedical samples at larger and larger scale. However, even in cleared samples, multiple light scattering often degrades the imaging contrast and widens the optical sectioning. Accumulation of scattering intensifies these negative effects as light propagates inside the tissue, which accentuates the issues when imaging large samples. With axially swept light-sheet microscopy (ASLM), centimeter-scale samples can be scanned with a uniform micrometric optical sectioning. But to fully utilize these benefits for 3D-imaging in biomedical tissue samples, suppression of scattered light is needed. Here, we address this by merging ASLM with light-sheet based structured illumination into Structured Illumination Light-sheet Microscopy with Axial Sweeping (SILMAS). The SILMAS method thus enables high-contrast imaging, isotropic micrometric resolution and uniform optical sectioning in centimeter-scale scattering samples, creating isotropic 3D-volumes of e.g., whole mouse brains without the need for any computation-heavy post-processing. We demonstrate the effectiveness of the approach in agarose gel phantoms with fluorescent beads, and in an PFF injected alpha-synuclein transgenic mouse model tagged with a green fluorescent protein (SynGFP). SILMAS imaging is compared to standard ASLM imaging on the same samples and using the same optical setup, and is shown to increase contrast by as much as 370% and reduce widening of optical sectioning by 74%. With these results, we show that SILMAS improves upon the performance of current state-of-the-art light-sheet microscopes for large and imperfectly cleared tissue samples and is a valuable addition to the LSFM family.

Biochemistry of the anterior, medial, and posterior genioglossus in the aged rat.

Age-related tongue weakness may contribute to swallowing deficits in the elderly. One contributing factor may be an alteration in muscle-fiber-type properties with aging. However, it is not clear how muscle fiber types within the aged tongue may vary from those found in young adults, or how fiber types may vary across the anteroposterior axis of the extrinsic tongue muscles. We examined the myosin heavy chain (MHC) composition of anterior, medial, and posterior sections of the genioglossus muscle (GG) in ten old male Fischer 344/Brown Norway rats and compared findings to previously reported data from young adult male rats. Significant differences (p < 0.01) between young adult and old rats were found in the distribution of MHC isoforms along the anteroposterior axis of the muscle. In the anterior, medial, and posterior regions, there was a significantly smaller proportion of type IIb MHC in the old rat GG muscles, while the proportion of type IIx MHC was significantly greater. In the medial region, the proportion of type I MHC was found to be significantly greater in the old rats. Thus, we found a shift to more slowly contracting muscle fibers in the aged rat tongue.

Trans-synaptic and retrograde axonal spread of Lewy pathology following pre-formed fibril injection in an in vivo A53T alpha-synuclein mouse model of synucleinopathy.

It is necessary to develop an understanding of the specific mechanisms involved in alpha-synuclein aggregation and propagation to develop disease modifying therapies for age-related synucleinopathies, including Parkinson's disease and Dementia with Lewy Bodies. To adequately address this question, we developed a new transgenic mouse model of synucleinopathy that expresses human A53T SynGFP under control of the mouse prion protein promoter. Our characterization of this mouse line demonstrates that it exhibits several distinct advantages over other, currently available, mouse models. This new model allows rigorous study of the initial location of Lewy pathology formation and propagation in the living brain, and strongly suggests that aggregation begins in axonal structures with retrograde propagation to the cell body. This model also shows expeditious development of alpha-synuclein pathology following induction with small, in vitro-generated alpha-synuclein pre-formed fibrils (PFFs), as well as accelerated cell death of inclusion-bearing cells. Using this model, we found that aggregated alpha-synuclein somatic inclusions developed first in neurons, but later showed a second wave of inclusion formation in astrocytes. Interestingly, astrocytes appear to survive much longer after inclusion formation than their neuronal counterparts. This model also allowed careful study of peripheral-to-central spread of Lewy pathology after PFF injection into the hind limb musculature. Our results clearly show evidence of progressive, retrograde trans-synaptic spread of Lewy pathology through known neuroanatomically connected pathways in the motor system. As such, we have developed a promising tool to understand the biology of neurodegeneration associated with alpha-synuclein aggregation and to discover new treatments capable of altering the neurodegenerative disease course of synucleinopathies.

Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Cross-activation and detraining effects of tongue exercise in aged rats.

Voice and swallowing deficits can occur with aging. Tongue exercise paired with a swallow may be used to treat swallowing disorders, but may also benefit vocal function due to cross-system activation effects. It is unknown how exercise-based neuroplasticity contributes to behavior and maintenance following treatment. Eighty rats were used to examine behavioral parameters and changes in neurotrophins after tongue exercise paired with a swallow. Tongue forces and ultrasonic vocalizations were recorded before and after training/detraining in young and old rats. Tissue was analyzed for neurotrophin content. Results showed tongue exercise paired with a swallow was associated with increased tongue forces at all ages. Gains diminished after detraining in old rats. Age-related changes in vocalizations, neurotrophin 4 (NT4), and brain derived neurotrophic factor (BDNF) were found. Minimal cross-system activation effects were observed. Neuroplastic benefits were demonstrated with exercise in old rats through behavioral improvements and up-regulation of BDNF in the hypoglossal nucleus. Tongue exercise paired with a swallow should be developed, studied, and optimized in human clinical research to treat swallowing and voice disorders in elderly people.

Exercise-induced rescue of tongue function without striatal dopamine sparing in a rat neurotoxin model of Parkinson disease.

Unilateral lesions to the medial forebrain bundle with 6-hydroxydopamine (6-OHDA) lead to force and timing deficits during a complex licking task. We hypothesized that training targeting tongue force generation during licking would improve timing and force measures and also lead to striatal dopamine sparing. Nine month-old male Fisher344/Brown Norway rats were used in this experiment. Sixteen rats were in the control condition and received tongue exercise (n=8) or no exercise (n=8). Fourteen rats were in the 6-OHDA lesion condition and underwent tongue exercise (n=7) and or no exercise (n=7). Following 4 weeks of training and post-training measures, all animals underwent bilateral stimulation of the hypoglossal nerves to measure muscle contractile properties and were then transcardially perfused and brain tissues collected for immunohistochemistry to examine striatal dopamine content. Results demonstrated that exercise animals performed better for maximal force, average force, and press rate than their no-exercise counterparts, and the 6-OHDA animals that underwent exercise performed as well as the Control No Exercise group. Interestingly, there were no group differences for tetanic muscle force, despite behavioral recovery of forces. Additionally, behavioral and neurochemical analyses indicate that there were no differences in striatal dopamine. Thus, targeted exercise can improve tongue force and timing deficits related to 6-OHDA lesions and this exercise likely has a central, versus peripheral (muscle strength) mechanism. However, this mechanism is not related to sparing of striatal dopamine content.

The effect of age and tongue exercise on BDNF and TrkB in the hypoglossal nucleus of rats.

Age-associated changes in tongue musculature may contribute to dysphagia. One possible treatment is tongue exercise. Exercise induces synaptic plasticity by increasing neurotrophic factors in spinal cord and limb musculature. However, effects of exercise on neurotrophic factors in the cranial sensorimotor system are unknown. Our purpose was to examine the effects of age and exercise on brain-derived neurotrophic factor (BDNF) and its receptor TrkB in the rat hypoglossal nucleus. Young, middle-aged, and old rats were assigned to exercise or no-exercise control conditions. Exercise animals were trained to perform a tongue press task for 8 weeks. Samples from the hypoglossal nucleus were analyzed for BDNF and TrkB immunoreactivity. Baseline maximum tongue forces were similar in all age groups and increased significantly following exercise. BDNF immunoreactivity did not show a significant decrease with age in control group. However, in the exercise group, BDNF was significantly increased in young animals. TrkB immunoreactivity decreased significantly with age in control group, but did not change with exercise. BDNF and TrkB immunoreactivity levels were positively correlated with exercise in young and middle aged animals, but were negatively or weakly correlated with exercise in old animals and with a lack of exercise in no-exercise controls. Tongue exercise was associated with increased tongue forces in rats at all ages. While increases in BDNF and TrkB levels associated with exercise may play a role in mechanisms contributing to increased tongue forces in young and middle-aged rats, other mechanisms may be involved in increased tongue forces observed in old rats.

Tongue force and timing deficits in a rat model of Parkinson disease.

Deficits in tongue function in conjunction with airway compromise can contribute to dysphagia associated with Parkinson disease (PD). However, it is unknown if these deficits are related to the primary disease pathology in PD, nigrostriatal dopamine depletion. To directly study the impact of striatal dopamine depletion on tongue function, we used unilateral infusion of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle and measured tongue force and timing parameters during a complex tongue protrusion task for a water reward. Maximal and average forces were significantly diminished and average press time was significantly longer after neurotoxin administration, reflecting aspects of bradykinesia and hypokinesia associated with PD. Our findings suggest that even unilateral deficits to the nigrostriatal dopamine system may be contributing to some of the lingual sensorimotor deficits seen in PD. Because previous research in rat models of PD has shown that targeted training of the limb can rescue behavioral deficits and spare striatal dopamine neurons, early intervention for cranial sensorimotor deficits may also be indicated.