Alterations of intrinsic tongue muscle properties with aging.

INTRODUCTION: Age-related decline in the intrinsic lingual musculature could contribute to swallowing disorders, yet the effects of age on these muscles is unknown. We hypothesized there is reduced muscle fiber size and shifts to slower myosin heavy chain (MyHC) fiber types with age. METHODS: Intrinsic lingual muscles were sampled from 8 young adult (9 months) and 8 old (32 months) Fischer 344/Brown Norway rats. Fiber size and MyHC were determined by fluorescent immunohistochemistry. RESULTS: Age was associated with a reduced number of rapidly contracting muscle fibers, and more slowly contracting fibers. Decreased fiber size was found only in the transverse and verticalis muscles. DISCUSSION: Shifts in muscle composition from faster to slower MyHC fiber types may contribute to age-related changes in swallowing duration. Decreasing muscle fiber size in the protrusive transverse and verticalis muscles may contribute to reductions in maximum isometric tongue pressure found with age. Differences among regions and muscles may be associated with different functional demands. Muscle Nerve 56: E119-E125, 2017.

Preparing the periphery for a subsequent behavior: motor neuronal activity during biting generates little force but prepares a retractor muscle to generate larger forces during swallowing in Aplysia.

Some behaviors occur in obligatory sequence, such as reaching before grasping an object. Can the earlier behavior serve to prepare the musculature for the later behavior? If it does, what is the underlying neural mechanism of the preparation? To address this question, we examined two feeding behaviors in the marine mollusk Aplysia californica, one of which must precede the second: biting and swallowing. Biting is an attempt to grasp food. When that attempt is successful, the animal immediately switches to swallowing to ingest food. The main muscle responsible for pulling food into the buccal cavity during swallowing is the I3 muscle, whose motor neurons B6, B9, and B3 have been previously identified. By performing recordings from these neurons in vivo in intact, behaving animals or in vitro in a suspended buccal mass preparation, we demonstrated that the frequencies and durations of these motor neurons increased from biting to swallowing. Using the physiological patterns of activation to drive these neurons intracellularly, we further demonstrated that activating them using biting-like frequencies and durations, either alone or in combination, generated little or no force in the I3 muscle. When biting-like patterns preceded swallowing-like patterns, however, the forces during the subsequent swallowing-like patterns were significantly enhanced. Sequences of swallowing-like patterns, either with these neurons alone or in combination, further enhanced forces in the I3 muscle. These results suggest a novel mechanism for enhancing force production in a muscle, and may be relevant to understanding motor control in vertebrates.

Motor neuronal activity varies least among individuals when it matters most for behavior.

How does motor neuronal variability affect behavior? To explore this question, we quantified activity of multiple individual identified motor neurons mediating biting and swallowing in intact, behaving Aplysia californica by recording from the protractor muscle and the three nerves containing the majority of motor neurons controlling the feeding musculature. We measured multiple motor components: duration of the activity of identified motor neurons as well as their relative timing. At the same time, we measured behavioral efficacy: amplitude of grasping movement during biting and amplitude of net inward food movement during swallowing. We observed that the total duration of the behaviors varied: Within animals, biting duration shortened from the first to the second and third bites; between animals, biting and swallowing durations varied. To study other sources of variation, motor components were divided by behavior duration (i.e., normalized). Even after normalization, distributions of motor component durations could distinguish animals as unique individuals. However, the degree to which a motor component varied among individuals depended on the role of that motor component in a behavior. Motor neuronal activity that was essential for the expression of biting or swallowing was similar among animals, whereas motor neuronal activity that was not essential for that behavior varied more from individual to individual. These results suggest that motor neuronal activity that matters most for the expression of a particular behavior may vary least from individual to individual. Shaping individual variability to ensure behavioral efficacy may be a general principle for the operation of motor systems.

The significance of dynamical architecture for adaptive responses to mechanical loads during rhythmic behavior.

Many behaviors require reliably generating sequences of motor activity while adapting the activity to incoming sensory information. This process has often been conceptually explained as either fully dependent on sensory input (a chain reflex) or fully independent of sensory input (an idealized central pattern generator, or CPG), although the consensus of the field is that most neural pattern generators lie somewhere between these two extremes. Many mathematical models of neural pattern generators use limit cycles to generate the sequence of behaviors, but other models, such as a heteroclinic channel (an attracting chain of saddle points), have been suggested. To explore the range of intermediate behaviors between CPGs and chain reflexes, in this paper we describe a nominal model of swallowing in Aplysia californica. Depending upon the value of a single parameter, the model can transition from a generic limit cycle regime to a heteroclinic regime (where the trajectory slows as it passes near saddle points). We then study the behavior of the system in these two regimes and compare the behavior of the models with behavior recorded in the animal in vivo and in vitro. We show that while both pattern generators can generate similar behavior, the stable heteroclinic channel can better respond to changes in sensory input induced by load, and that the response matches the changes seen when a load is added in vivo. We then show that the underlying stable heteroclinic channel architecture exhibits dramatic slowing of activity when sensory and endogenous input is reduced, and show that similar slowing with removal of proprioception is seen in vitro. Finally, we show that the distributions of burst lengths seen in vivo are better matched by the distribution expected from a system operating in the heteroclinic regime than that expected from a generic limit cycle. These observations suggest that generic limit cycle models may fail to capture key aspects of Aplysia feeding behavior, and that alternative architectures such as heteroclinic channels may provide better descriptions.

Differential activation of an identified motor neuron and neuromodulation provide Aplysia's retractor muscle an additional function.

To survive, animals must use the same peripheral structures to perform a variety of tasks. How does a nervous system employ one muscle to perform multiple functions? We addressed this question through work on the I3 jaw muscle of the marine mollusk Aplysia californica's feeding system. This muscle mediates retraction of Aplysia's food grasper in multiple feeding responses and is innervated by a pool of identified neurons that activate different muscle regions. One I3 motor neuron, B38, is active in the protraction phase, rather than the retraction phase, suggesting the muscle has an additional function. We used intracellular, extracellular, and muscle force recordings in several in vitro preparations as well as recordings of nerve and muscle activity from intact, behaving animals to characterize B38's activation of the muscle and its activity in different behavior types. We show that B38 specifically activates the anterior region of I3 and is specifically recruited during one behavior, swallowing. The function of this protraction-phase jaw muscle contraction is to hold food; thus the I3 muscle has an additional function beyond mediating retraction. We additionally show that B38's typical activity during in vivo swallowing is insufficient to generate force in an unmodulated muscle and that intrinsic and extrinsic modulation shift the force-frequency relationship to allow contraction. Using methods that traverse levels from individual neuron to muscle to intact animal, we show how regional muscle activation, differential motor neuron recruitment, and neuromodulation are key components in Aplysia's generation of multifunctionality.

Differential impact of unilateral stroke on the bihemispheric motor cortex representation of the jaw and tongue muscles in young and aged rats.

Introduction: Dysphagia commonly occurs after stroke, yet the mechanisms of post-stroke corticobulbar plasticity are not well understood. While cortical activity associated with swallowing actions is bihemispheric, prior research has suggested that plasticity of the intact cortex may drive recovery of swallowing after unilateral stroke. Age may be an important factor as it is an independent predictor of dysphagia after stroke and neuroplasticity may be reduced with age. Based on previous clinical studies, we hypothesized that cranial muscle activating volumes may be expanded in the intact hemisphere and would contribute to swallowing function. We also hypothesized that older age would be associated with limited map expansion and reduced function. As such, our goal was to determine the impact of stroke and age on corticobulbar plasticity by examining the jaw and tongue muscle activating volumes within the bilateral sensorimotor cortices. Methods: Using the middle cerebral artery occlusion rat stroke model, intracortical microstimulation (ICMS) was used to map regions of sensorimotor cortex that activate tongue and jaw muscles in both hemispheres. Young adult (7 months) and aged (30 months) male F344 × BN rats underwent a stroke or sham-control surgery, followed by ICMS mapping 8 weeks later. Videofluoroscopy was used to assess oral-motor functions. Results: Increased activating volume of the sensorimotor cortex within the intact hemisphere was found only for jaw muscles, whereas significant stroke-related differences in tongue activating cortical volume were limited to the infarcted hemisphere. These stroke-related differences were correlated with infarct size, such that larger infarcts were associated with increased jaw representation in the intact hemisphere and decreased tongue representation in the infarcted hemisphere. We found that both age and stroke were independently associated with swallowing differences, weight loss, and increased corticomotor thresholds. Laterality of tongue and jaw representations in the sham-control group revealed variability between individuals and between muscles within individuals. Conclusion: Our findings suggest the role of the intact and infarcted hemispheres in the recovery of oral motor function may differ between the tongue and jaw muscles, which may have important implications for rehabilitation, especially hemisphere-specific neuromodulatory approaches. This study addressed the natural course of recovery after stroke; future work should expand to focus on rehabilitation.

Changes in ultrasonic vocalizations after unilateral cerebral ischemia in a rat stroke model.

Stroke frequently results in communication impairments that negatively impact quality of life and overall recovery, yet the biological mechanisms underlying these changes are not well understood. Ultrasonic vocalizations (USVs) in rodent models of disease and aging have been used to improve our understanding of the biological mechanisms that underlie vocal deficits and their response to interventions. Changes in USVs after middle cerebral artery occlusion (MCAO) in mice have been reported, yet rat models have significant anatomical and behavioral advantages over mice, including the ability to vocally train rats with an established paradigm. We sought to determine whether a unilateral MCAO rat stroke model provides a biologically and behaviorally relevant way to study post stroke vocalization deficits. We hypothesized that left MCAO would be associated with changes in USVs. Six weeks after MCAO or sham-control surgery, USVs were recorded in rats using an established mating paradigm. Stroke was associated with differences in USV acoustics including more frequent use of simple calls characterized by shorter durations and restricted bandwidths. These parameters were also found to correlate with post stroke lingual weakness. This is the first study to describe changes to rat USVs using a stroke model. These results suggest the unilateral MCAO rat stroke model is a biologically and behaviorally relevant model to understand how stroke affects vocal behaviors.

Central activation deficits contribute to post stroke lingual weakness in a rat model.

Lingual weakness frequently occurs after stroke and is associated with deficits in speaking and swallowing. Chronic weakness after stroke has been attributed to both impaired central activation of target muscles and reduced force-generating capacity within muscles. How these factors contribute to lingual weakness is not known. We hypothesized that lingual weakness due to middle cerebral artery occlusion (MCAO) would manifest as reduced muscle force capacity and reduced muscle activation. Rats were randomized into MCAO or sham surgery groups. Maximum volitional tongue forces were quantified 8 wk after surgery. Hypoglossal nerve stimulation was used to assess maximum stimulated force, muscle twitch properties, and force-frequency response. The central activation ratio was determined by maximum volitional/maximum stimulated force. Genioglossus muscle fiber type properties and neuromuscular junction innervation were assessed. Maximum volitional force and the central activation ratio were significantly reduced with MCAO. Maximum stimulated force was not significantly different. No significant differences were found for muscle twitch properties, unilateral contractile properties, muscle fiber type percentages, or fiber size. However, the twitch/tetanus ratio was significantly increased in the MCAO group relative to sham. A small but significant increase in denervated neuromuscular junctions (NMJs) and fiber-type grouping occurred in the contralesional genioglossus. Results suggest that the primary cause of chronic lingual weakness after stroke is impaired muscle activation rather than a deficit of force-generating capacity in lingual muscles. Increased fiber type grouping and denervated NMJs in the contralesional genioglossus suggest that partial reinnervation of muscle fibers may have preserved force-generating capacity, but not optimal activation patterns.NEW & NOTEWORTHY Despite significant reductions in maximum volitional forces, the intrinsic force-generating capacity of the protrusive lingual muscles was not reduced with unilateral cerebral ischemia. Small yet significant increases in denervated NMJs and fiber-type grouping of the contralesional genioglossus suggest that the muscle underwent denervation and reinnervation. Together these results suggest that spontaneous neuromuscular plasticity was sufficient to prevent atrophy, yet central activation deficits remain and contribute to chronic lingual weakness after stroke.

Reduced tongue force and functional swallowing changes in a rat model of post stroke dysphagia.

PURPOSE: Dysphagia is a common problem after stroke that is often associated with tongue weakness. However, the physiological mechanisms of post-stroke tongue muscle weakness and optimal treatments have not been established. To advance understanding of physiological mechanisms of post stroke dysphagia, we sought to validate the unilateral transient middle cerebral artery occlusion (MCAO) rat model of ischemic stroke as a translational model of post stroke dysphagia. Our goal was to establish clinically relevant measures and chronicity of functional deficits; criteria that increase the likelihood that findings will translate to the clinic. We hypothesized that MCAO would cause tongue weakness and functional swallowing changes. METHODS: Maximum voluntary tongue forces and videofluoroscopic swallowing studies were collected in 8-week old male Sprague-Dawley rats prior to receiving either a left MCAO (N = 10) or sham (N = 10) surgery. Tongue forces and VFSS were reassessed at 1 and 8 weeks post-surgery. RESULTS: Maximum voluntary tongue force, bolus area, and bolus speed were significantly reduced in the MCAO group at the 1 and 8-week timepoints. CONCLUSION: Clinically relevant changes to swallowing and tongue force support the use of the MCAO rat model as a translational model of post stroke dysphagia. This model will allow for future studies to improve our understanding of the physiology contributing to these functional changes as well as the impact of therapeutic interventions on physiological targets and function.

Age-related alterations in swallowing biomechanics.

BACKGROUND: Aging rodent models allow for the discovery of underlying mechanisms of cranial muscle dysfunction. Methods are needed to allow quantification of complex, multivariate biomechanical movements during swallowing. Videofluoroscopic swallow studies (VSS) are the standard of care in assessment of swallowing disorders in patients and validated quantitative, kinematic, and morphometric analysis methods have been developed. Our purpose was to adapt validated morphometric techniques to the rodent to computationally analyze swallowing dysfunction in the aging rodent. METHODS: VSS, quantitative analyses (bolus area, bolus velocity, mastication rate) and a rodent specific multivariate, morphometric computational analysis of swallowing biomechanics were performed on 20 swallows from 5 young adult and 5 old Fischer 344/Brown Norway rats. Eight anatomical landmarks were used to track the relative change in position of skeletal levers (cranial base, vertebral column, mandible) and soft tissue landmarks (upper esophageal sphincter, base of tongue). RESULTS: Bolus area significantly increased and mastication rate significantly decreased with age. Aging accounted for 77.1% of the variance in swallow biomechanics, and 18.7% of the variance was associated with swallow phase (oral vs pharyngeal). Post hoc analyses identified age-related alterations in tongue base retraction, mastication, and head posture during the swallow. CONCLUSION: Geometric morphometric analysis of rodent swallows suggests that swallow biomechanics are altered with age. When used in combination with biological assays of age-related adaptations in neuromuscular systems, this multivariate analysis may increase our understanding of underlying musculoskeletal dysfunction that contributes to swallowing disorders with aging.

Tongue Force Training Induces Plasticity of the Lingual Motor Cortex in Young Adult and Aged Rats.

Tongue exercise programs are used clinically for dysphagia in aged individuals and have been shown to improve lingual strength. However, the neural mechanisms of age-related decline in swallowing function and its association with lingual strength are not well understood. Using an established rat model of aging and tongue exercise, we hypothesized that the motor cortex of aged rats would have a smaller lingual motor map area than young adult rats and would increase in size as a function of tongue exercise. Over 8 weeks, rats either underwent a progressive resistance tongue exercise program (TE), learned the task but did not exercise (trained controls, TC), or were naïve untrained controls (UC). Cortical motor map areas for tongue and jaw were determined using intracortical microstimulation (ICMS). Rats in the TE and TC groups had a significantly larger motor cortex region for the tongue than the UC group. Lingual cortical motor area was not correlated with protrusive tongue force gains and did not differ significantly with age. These results suggest that learning a novel tongue force skill was sufficient to induce plasticity of the lingual motor cortex yet increasing tongue strength with progressive resistance exercise did not significantly expand the lingual motor area beyond the gains that occurred through the skilled learning component.

Differential impact of tongue exercise on intrinsic lingual muscles.

OBJECTIVES/HYPOTHESIS: Aging is associated with muscle fiber hypotrophy and decreased percentages of rapidly contracting myosin heavy chain (MyHC) type IIb muscle fibers. Tongue exercise programs used to treat dysphagia target age-related decline in tongue muscle function, but the impact of exercise on the intrinsic tongue muscles is unknown. We hypothesized that exercise would induce muscle fiber hypertrophy and increase the percentage of MyHC IIa fibers in the intrinsic tongue. STUDY DESIGN: Animal model. METHODS: Eight old and eight young-adult rats underwent 8 weeks of tongue exercise training, and 8 animals of each age group served as controls. Longitudinal, transverse, and verticalis muscle samples from the anterior, middle, and posterior regions of the tongue were sectioned and stained to determine muscle fiber diameter and MyHC composition. RESULTS: MyHC fiber type distribution was altered by exercise, and the effects differed by muscle and region of the tongue. In the exercise groups, as compared to the control groups, the anterior transverse and middle superior longitudinal muscles had significantly reduced percentages of MyHC IIx positive fibers and higher percentages of rapidly contracting fatigable MyHC IIb positive muscle fibers, whereas the middle transverse and posterior longitudinal muscles had increased percentages of the less rapidly contracting and more fatigue-resistant MyHC IIa fibers. The impact of exercise did not differ with age, as there was no significant interaction between age and exercise. Tongue exercise had no significant effect on muscle fiber diameter. CONCLUSIONS: The impact of exercise varied among the tongue muscles, which may indicate different functional contributions to the tongue exercise task. LEVEL OF EVIDENCE: NA Laryngoscope, 128:2245-2251, 2018.

Sensory Feedback Reduces Individuality by Increasing Variability within Subjects.

Behavioral variability is ubiquitous [1-6], yet variability is more than just noise. Indeed, humans exploit their individual motor variability to improve tracing and reaching tasks [7]. What controls motor variability? Increasing the variability of sensory input, or applying force perturbations during a task, increases task variability [8, 9]. Sensory feedback may also increase task-irrelevant variability [9, 10]. In contrast, sensory feedback during locust flight or to multiple cortical areas just prior to task performance decreases variability during task-relevant motor behavior [11, 12]. Thus, how sensory feedback affects both task-relevant and task-irrelevant motor outputs must be understood. Furthermore, since motor control is studied in populations, the effects of sensory feedback on variability must also be understood within and across subjects. For example, during locomotion, each step may vary within and across individuals, even when behavior is normalized by step cycle duration [13]. Our previous work demonstrated that motor components that matter for effective behavior show less individuality [14]. Is sensory feedback the mechanism for reducing individuality? We analyzed durations and relative timings of motor pools within swallowing motor patterns in the presence and absence of sensory feedback and related these motor program components to behavior. Here, at the level of identified motor neurons, we show that sensory feedback to motor program components highly correlated with behavioral efficacy reduces variability across subjects but-surprisingly-increases variability within subjects. By controlling intrinsic, individual differences in motor neuronal activity, sensory feedback provides each subject access to a common solution space.

Electrode fabrication and implantation in Aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.