Tongue Protrudor Research Articles

Distribution of muscarinic acetylcholine receptors in hypoglossal motoneurons across postnatal maturation

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. During REM sleep, hypoglossal motoneurons (XII MNs), which innervate genioglossus, receive less excitatory noradrenergic drive and may be inhibited by activation of muscarinic acetylcholine receptors. Data from intact adult rats during natural sleep implicates an inhibitory effect of muscarinic acetylcholine receptors on XII MNs. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at XII MNs. Our project examines the changes to distribution of muscarinic acetylcholine receptors (mAChRs) at XII MNs across postnatal maturation. We hypothesize there is an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) mAChRs at XII MNs with postnatal maturation. We performed double-labeled immunofluorescence experiments on perfused transverse brainstem slices (20μm) under identical conditions across six postnatal age groups: P0-P2, P3-P5, P6-P10, P11-P13, P14-P17, and adult against mAChR targets M1, M2, M3, M5. Slices were co-labeled with choline acetyltransferase (ChAT) and 4’,6-diamidino-2-phenylindole (DAPI). Images were collected using a confocal microscope. 10 individual XII MNs were identified in each slice using positive ChAT labeling as well as anatomical location. ImageJ was used to determine the average XII MN mAChR subtype intensity. Preliminary data (n = 3) indicate that M1 receptors showed a transitory increase in labeling intensity in XII MNs followed by a decrease across development (P1=89±11%, P4-5=100%, P9=76±18%, P12-13=67±8%, P17=67±4%, adult=56±7%, p = 0.13). M3 (n = 3) showed a trend for a transient increase in labeling intensity at P17 (P1=78±41%, P4-5=87±3%, P9=61±13%, P12-13=86±34%, P17=100%, adult=80±23, p = 0.60). M5 (n = 3) expression intensity decreased consistently over maturation (P1=100%, P4-5=92±34%, P9=75±23%, P12-13=70±10%, P17=66±14%, adult=39±12%, p = 0.0257). In contrast, M2 (n = 3) receptor expression intensity remained relatively consistent across maturation (P1 = 88±23%, P4-5 = 100%, P9=88±10%, P12-13 = 75±15%, P17 = 86±18%, adult = 75±10%). These data partly support our hypothesis of a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors across maturation. The decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift in muscarinic modulation from excitation to inhibition with postnatal maturation. Arizona Alzheimer's Consortium, NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Articulation differences of /s/ observed using inverse tongue atlas modeling

Depending on the vowel context, the consonant /s/ can be articulated by engaging different functional units of the tongue. An inverse finite element (FE) tongue model comprising hexahedral elements generated from a 4D statistical MRI atlas of 22 speakers performing the speech tasks “a geese” (tongue moving forwards) and “a souk” (tongue moving backwards) is used to study this articulatory behavior. The model uses a state-of-the-art inverse tracking controller to simulate the motion of internal tissue points of the different speakers deformed into the atlas space. Motion tracking is successfully carried out by minimizing the L2-norm of velocity error of FEM nodes using the Cottle–Dantzig algorithm. The results show that for “a-geese,” the utterance of /s/ in context of vowel /i/ showed increase in activation of tongue protruder muscles such as genioglossus posterior, floor muscle geniohyoid by ∼5% more than in the case of “a-souk.” In “a-souk” relative activity increased in the tongue retractor muscles mid- and anterior-genioglossus, and superior longitudinal by ∼4%. Our findings are consistent with subject-specific state-of-the-art models and with articulatory expectations. The inverse atlas tongue model can be further used to estimate such articulation behavior on extended datasets of subjects performing more variations of speech tasks.

Oxytocin mediated excitation of hypoglossal motoneurons: implications for treating obstructive sleep apnea.

Clinical studies have shown that oxytocin administered intranasally (IN) decreased the incidence and duration of obstructive events in patients with obstructive sleep apnea (OSA). Although the mechanisms by which oxytocin promotes these beneficial effects are unknown, one possible target of oxytocin could be the excitation of tongue-projecting hypoglossal motoneurons in the medulla, that exert central control of upper airway patency. This study tested the hypothesis that IN oxytocin enhances tongue muscle activity via the excitation of hypoglossal motoneurons projecting to tongue protrudor muscles (PMNs). To test this hypothesis we performed in vivo and in vitro electrophysiological studies in C57BL6/J mice as well as fluorescent imaging studies in transgenic mice in which neurons that express oxytocin receptors co-express fluorescent protein. IN oxytocin significantly increased the amplitude of inspiratory-related tongue muscle activity. This effect was abolished by severing the medial branch of hypoglossal nerve that innervates PMNs of the tongue. Oxytocin receptor-positive neurons were more prevalent in the population of PMNs than in retractor-projecting hypoglossal motoneurons (RMNs). Oxytocin administration increased action potential firing in PMNs, but had no significant effect on firing activity in RMNs. In conclusion, IN oxytocin stimulates respiratory-relating tongue muscle activity likely acting on central hypoglossal motoneurons that provide tongue protrusion and upper airway opening. This mechanism may play a role in oxytocin-induced reductions in upper airway obstructions in patients with OSA.

Abstract 11871: Hypoglossal Nerve Stimulation as a Treatment for Sleep Apnea Does Not Lower Blood Pressure

Introduction: Obstructive sleep apnea (OSA) effects up to 30% of men and 15% of women in North America. Untreated, OSA is associated with the development of hypertension, heart failure, coronary artery disease, cardiac arrhythmia, and stroke. Treatment reduces these risks. The American Academy of Sleep Medicine outlines the first line treatment of OSA as continuous positive airway pressure (CPAP). However, many patients are unable to tolerate CPAP. One of the newest therapies approved in 2014 is implantation of a hypoglossal nerve stimulator (HNS) which functions by stimulating the tongue protrudor muscles to reduce upper airway obstruction. However, there is minimal research investigating HNS's effects on comorbidities. Currently there are two published studies showing improved blood pressure control and heart rate variability, but each study only included 46 patients. Several factors warrant a comparison including the lack of data, procedural risks, and procedural cost; especially, when considering the proven efficacy and low cost of CPAP. We present a retrospective analysis of blood pressure after HNS implantation. Hypothesis: We hypothesize that HNS implantation will improve blood pressure control. Methods: All cases of HNS implantation at MercyOne North Iowa Mason City, Iowa were retrospectively reviewed. Each case was manually reviewed for pre- and post-surgery blood pressure, heart failure exacerbations, arrhythmia, hemoglobin A1c, stroke, and acute coronary syndrome. Results: Fifty-nine patients had a mean change in blood pressure of +1.76 systolic and +0.76 diastolic with a standard error of 2.10 and 1.29 respectively. The mean follow-up time was 5.7 months after the fine-tuning sleep titration study. Five patients needed one less blood pressure medication and 6 patients needed one more. Preliminary regression analysis does not support gender, age, apnea-hypopnea index, or body mass index being predictive of change in blood pressure with HNS implantation. There was no statistically significant effect on other comorbidities. Conclusion: This study showed no clinically significant change in blood pressure after HNS implantation. Limitations include the small sample size and only one follow-up blood pressure.

Effects of postnatal maturation on muscarinic acetylcholine receptor distribution in hypoglossal motorneurons

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. Hypoglossal motoneurons (XII MNs), which innervate the genioglossus muscle, receive less excitatory noradrenergic drive, and may be inhibited by activation of muscarinic acetylcholine receptors, during REM sleep. Indeed, preliminary data from intact adult rats during natural sleep implicated an inhibitory effect of muscarinic acetylcholine receptors on hypoglossal motoneurons. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at hypoglossal motoneurons. Our project examines the distribution of muscarinic acetylcholine receptors at XII MNs, and how that changes across postnatal maturation. We hypothesizes there would be an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) muscarinic receptors in XII MNs with postnatal maturation. We performed double‐labeled immunofluorescence experiments on perfused 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0‐P2, P3‐P5, P6‐P10, P11‐P13, P14‐P17, and adult against muscarinic targets M1, M2, M3, M5. Slices were co‐labeled with choline acetyltransferase (ChAT) and 4’,6‐diamidino‐2‐phenylindole (DAPI). Images of the hypoglossal motor nucleus (HMN) were collected using a confocal microscope. ImageJ was used to determine the average HMN muscarine receptor subtype intensity across postnatal maturation. Preliminary data (n = 1) indicate that M1 receptors showed a consistent decrease in labeling intensity in the HMN across development (P5=100%, P13=80%, P17=67%, adult = 40%), whereas M3 (n = 1) (P4= 90%, P9= 73%, P13=89%, P17= 100%, adult= 41%) showed a transitory increases in expression before a final decrease into adulthood. M5 (n = 1 or 2) expression shows a bimodal distribution of labeling intensity (P0‐2= 100%, P4‐6= 58‐99%, P7‐10 = 52‐70%, P11‐13= 35‐58 %, P14‐17 = 27 % adult= 17‐37 %). In contrast, M2 receptor expression intensity remained relatively high with small fluctuations into adulthood (n=2), (P0‐2 = 92‐94 %, P4‐6 = 100%, P7‐10 = 92‐95 %, P11‐13 = 78‐107 %, P14‐17 = 88‐107 %, adult = 73‐77 %). These data partly support our hypothesis that there would be a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors. We speculate that the decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift of muscarinic modulation from excitation to inhibition with postnatal maturation.

Progressive Protrusive Tongue Exercise Does Not Alter Aging Effects in Retrusive Tongue Muscles.

Purpose: Exercise-based treatment approaches for dysphagia may improve swallow function in part by inducing adaptive changes to muscles involved in swallowing and deglutition. We have previously shown that both aging and progressive resistance tongue exercise, in a rat model, can induce biological changes in the genioglossus (GG); a muscle that elevates and protrudes the tongue. However, the impacts of progressive resistance tongue exercise on the retrusive muscles (styloglossus, SG; hyoglossus, HG) of the tongue are unknown. The purpose of this study was to examine the impact of a progressive resistance tongue exercise regimen on the retrusive tongue musculature in the context of aging. Given that aging alters retrusive tongue muscles to more slowly contracting fiber types, we hypothesized that these biological changes may be mitigated by tongue exercise.Methods: Hyoglossus (HG) and styloglossus (SG) muscles of male Fischer 344/Brown Norway rats were assayed in age groups of young (9 months old, n = 24), middle-aged (24 months old, n = 23), and old (32 months old, n = 26), after receiving an 8-week period of either progressive resistance protrusive tongue exercise, or sham exercise conditions. Following exercise, HG and SG tongue muscle contractile properties were assessed in vivo. HG and SG muscles were then isolated and assayed to determine myosin heavy chain isoform (MyHC) composition.Results: Both retrusive tongue muscle contractile properties and MyHC profiles of the HG and SG muscles were significantly impacted by age, but were not significantly impacted by tongue exercise. Old rats had significantly longer retrusive tongue contraction times and longer decay times than young rats. Additionally, HG and SG muscles showed significant MyHC profile changes with age, in that old groups had slower MyHC profiles as compared to young groups. However, the exercise condition did not induce significant effects in any of the biological outcome measures.Conclusion: In a rat model of protrusive tongue exercise, aging induced significant changes in retrusive tongue muscles, and these age-induced changes were unaffected by the tongue exercise regimen. Collectively, results are compatible with the interpretation that protrusive tongue exercise does not induce changes to retrusive tongue muscle function.

The Role of Arginine Vasopressin in Modulating Inspiratory Bursting Behavior in the Hypoglossal Motor Nucleus in Neonatal Mice

Arginine vasopressin (AVP) is classically known as a hormone, but it also acts as a neurotransmitter that binds V1a receptors in the central nervous system. V1a receptors are expressed at high levels early in postnatal life in cranial and spinal motoneurons. While AVP receptor expression levels decrease in some cranial motoneuron populations with increasing developmental age, AVP receptor mRNA levels are expressed at high levels in the hypoglossal (XII) motor nucleus in adult mice. Recordings from individual XII motoneurons demonstrated that AVP has excitatory effects on young XII motoneurons, although the signaling pathways and effector ion channels remain to be fully elucidated. Additionally, previous research demonstrated that GABAergic disinhibition within the paraventricular nucleus of adult male rats, which has the effect of promoting AVP release, led to stimulation of breathing and increased diaphragm and genioglossus (innervated by XII, primary tongue protruder) muscle activity. Whether AVP acts directly at XII motoneurons to potentiate inspiratory bursting is unknown. Our aim was to characterize the effects of AVP on inspiratory bursting behavior at XII motoneurons. To test our hypothesis that AVP will potentiate inspiratory bursting behavior, we utilized an in vitro medullary brainstem slice (600 µM) preparation of neonatal (postnatal day, P0-5) CD1 mouse brains of either sex that contains the nuclei for respiratory movement of the tongue. By superfusing the slice with warmed artificial cerebral spinal fluid, we were able to record inspiratory bursting activity using a suction electrode under baseline conditions and in response to local drug application. Local injections of AVP at 0.01 µM had little effect on inspiratory bursting amplitude (111% ± 3% of baseline, n=6), but at 0.1 µM or 1 µM, initial results suggest it increased inspiratory burst amplitude (134% ± 5% of baseline, n=13 and 130% ± 8% of baseline, n=7 respectively, p = 0.0889). The excitatory effect of AVP was significantly diminished using a V1a receptor antagonist, d(CH2)51Tyr(Me)2Arg8) Vasopressin (500nM) (Initial AVP increase in burst amplitude: 141% ± 8%; AVP after antagonist: 113% ± 4%; AVP after washout: 120% ± 3%, n=10, p = 0.0437). Vasopressin and oxytocin are closely related nonapeptides, and since the XII motor nucleus also expresses oxytocin receptors, we next tested whether the AVP effects could be attributed to activation of the oxytocin receptor. Our preliminary results suggest that the oxytocin receptor antagonist, L-371,257 (50nM), did attenuate the response of AVP (Initial AVP increase in burst amplitude: 144% ± 11%; AVP after antagonist: 114% ± 5%; AVP after Washout: 122% ± 6%, n=8, p = 0.0901). Lastly, we discovered that vasopressin-related potentiation of inspiratory burst amplitude has an age-dependent increase from P0-5 (regression fit: y=13.933x+12.401, R2=0.4411, p=0.000402). Our results indicate that vasopressin, acting at V1a, and possibly oxytocin, receptors, can potentiate XII inspiratory bursting behavior in neonatal mice. Whether vasopressin has a role in maintaining airway patency in vivo in adult animals will need to be addressed in future studies.

Cholinergic Modulation by Activation of Muscarinic Acetylcholine Receptor Subtypes Modulates Inspiratory Bursting in Hypoglossal Motoneurons in Mice in Vitro

During wakefulness, tonic and respiratory input provides drive to upper airway dilatory muscles, and in particular genioglossus, the main tongue protruder, which stiffens the airway and keeps airway resistance low. These respiratory muscles experience decrements in activity during rapid eye movement sleep. Disruption in motor output and insufficient muscle tone of the tongue due to inhibition, or loss of excitation, of hypoglossal motoneurons (which innervate tongue muscles) contributes to sleep-disordered breathing. Recent data from adult rats in vivo suggest that muscarinic inhibition of hypoglossal motoneurons may be a major mechanism leading to loss of tongue muscle tone during sleep. However, available data indicate that there are contrasting effects of muscarinic modulation: in neonatal rhythmic slice preparations, muscarine applied within the hypoglossal nucleus has an excitatory effect on inspiratory burst amplitude. Muscarinic receptor subtypes can be broadly divided into those that are coupled to excitatory G-protein coupled receptors (M1, M3, M5) and those that are coupled to inhibitory G-protein coupled receptors (M2, M4). Hypoglossal motoneurons express M1, M2, M5, and possibly M3 receptor subtypes. Many modulatory systems undergo postnatal maturation, which we hypothesize underlies the switch in muscarinic modulation from net excitation to net inhibition of inspiratory bursting behavior at hypoglossal motoneurons. As an initial test of our hypothesis, our goal was to determine the contribution of excitatory and inhibitory muscarine receptor subtypes to the muscarinic modulation of inspiratory burst output from hypoglossal motoneurons in neonatal mice. Using the in vitro rhythmic slice preparation, we first investigated inspiratory behavior by observing the net effects of muscarine locally applied into the hypoglossal nucleus (100 mM, 30 s) on inspiratory burst amplitude in neonatal CD-1 mice (postnatal day, P0-P5). Our data confirm that in mice ages P0-5, local application of muscarine increased inspiratory burst amplitude (range: 53-180% potentiation, n = 9) compared to control inspiratory burst activity. Preliminary results suggest that the local application of M2 antagonist methoctramine in addition to muscarine (range: 20%-135% potentiation, n=4) had little effect on inspiratory behavior compared to baseline effects of muscarine (range: 4%-143% potentiation). Next, we tested the contribution of M3 receptors to muscarinic modulation on inspiratory burst amplitude. At baseline, locally applied muscarine increased inspiratory burst amplitude (range: 20%-63% potentiation); the muscarinic potentiation was attenuated during bath application of the M3 receptor-preferring antagonist 4-DAMP (range: 17%-25% potentiation) and recovered upon washout (range: 13%-117% potentiation, n=3). These preliminary results indicate muscarinic excitation is mediated in part by the M3-receptor subtype. Future experiments will determine how the M2 and M3 receptor contributions change with postnatal maturation.

Chronic intermittent hypoxia increases excitability and synaptic excitation of protrudor and retractor hypoglossal motoneurones.

Dysfunctions in the hypoglossal control of tongue extrinsic muscles are implicated in obstructive sleep apnoea (OSA) syndrome. Chronic intermittent hypoxia (CIH), an important feature of OSA syndrome, produces deleterious effects on the motor control of oropharyngeal resistance, but whether the hypoglossal motoneurones innervating the tongue extrinsic muscles are affected by CIH is unknown. We show that CIH enhanced the respiratory-related activity of rat hypoglossal nerve innervating the protrudor and retractor tongue extrinsic muscles. Intracellular recordings revealed increases in respiratory-related firing frequency and synaptic excitation of inspiratory protrudor and retractor hypoglossal motoneurones after CIH. CIH also increased their intrinsic excitability, depolarised resting membrane potential and reduced K+ -dominated leak conductance. CIH affected the breathing-related synaptic control and intrinsic electrophysiological properties of protrudor and retractor hypoglossal motoneurones to optimise the neural control of oropharyngeal function. Inspiratory-related tongue movements and oropharyngeal motor actions are controlled mainly by the protrudor and retractor extrinsic tongue muscles, which are innervated by the hypoglossal motoneurones. Chronic intermittent hypoxia (CIH), an important feature of obstructive sleep apnoea syndrome, produces detrimental effects on the contractile function of the tongue extrinsic muscles and the medullary inspiratory network of rodents. However, the impact of the CIH on the electrophysiological properties of protrudor and retractor hypoglossal motoneurones has not been described before. Using nerves and intracellular recordings in in situ preparation of rats (5weeks old), we tested the hypothesis that CIH (FiO2 of 0.06, SaO2 74%, during 30-40s, every 9min, 8h/day for 10days) increases the intrinsic excitability of protrudor and retractor motoneurones from the hypoglossal motor nucleus of rats. Recordings of hypoglossal nerve, before its bifurcation to innervate the tongue protrudor and retractor muscles, revealed that CIH enhances its pre-inspiratory, simultaneously with the presence of active expiration, and inspiratory activities. These changes were mediated by increases in the respiratory-related firing frequency and synaptic excitation of inspiratory protrudor and retractor hypoglossal motoneurones. Besides, CIH increases their intrinsic excitability and depolarises resting membrane potential by reducing a K+ -dominated leak conductance. In conclusion, CIH enhances the respiratory-related neural control of oropharyngeal function of rats by increasing the synaptic excitation, intrinsic excitability, and reducing leak conductance in both protrudor and retractor hypoglossal motoneurones. We propose that these network and cellular changes are important to optimise the oropharyngeal resistance in conditions related to intermittent hypoxia.

Characterization of effector ion channels that mediate excitatory cholinergic modulation of XII motoneurons in neonatal mice in vitro

Obstructive sleep apnea is a sleep disorder characterized by repeated stops and starts of breathing during sleep. During an apneic episode there is a decrease in upper airway muscle tone, particularly of the genioglossus muscle (primary tongue protruder). Recent data suggest that this sleep specific loss of muscle tone is due to muscarinic modulation of hypoglossal (XII) motoneurons that innervate genioglossus. In adult rats, during REM sleep, activation of muscarinic acetylcholine receptors depresses genioglossus inspiratory output. By contrast, in neonatal mouse brainstem slices, muscarine potentiates inspiratory output when locally applied to the XII motor nucleus. To determine whether these different effects of muscarine are due to postnatal maturation, we first need to determine the effector ion channels that mediate the excitatory response to muscarine in neonatal mice. In individual XII motoneurons recorded from neonatal brainstem slices, muscarinic receptor activation inhibited an apamin‐sensitive small conductance calcium dependent potassium current (SK) and potentiated a hyperpolarized‐activated mixed cation current (Ih). It was hypothesized that both effector ion channels contribute to the net potentiation of inspiratory drive by muscarine in neonatal mice. Therefore, we aimed to determine 1) the effect of inhibition of SK, and 2) the contribution of Ih modulation to the modulation of inspiratory drive by muscarinic acetylcholine receptor activation at XII motoneurons in neonatal mice. Using rhythmically medullary slice preparations from neonatal CD1 mice (postnatal day P0–5), we first investigated the effects of blocking SK on inspiratory burst output. Local application of apamin (30s) to the XII motor nucleus had a net effect of increasing inspiratory burst amplitude (n=3) to 134% of baseline (range 118–143%) or 132% (range 111–169%) of baseline (2 and 20 mM respectively). These data are important to establish the contributions of SK to inspiratory burst amplitude before testing how muscarine may modulate SK during inspiratory bursts. We then confirmed that muscarine had a net excitatory effect on inspiratory burst amplitude in neonatal mice (128 ± 6% of baseline, n = 9). Next, we aimed to investigate if muscarinic excitation is mediated by blocking SK channels. In one P3 preparation, local application of apamin attenuated the muscarinic‐mediated increase in XII inspiratory burst amplitude from 157% to 137%. Lastly, in one P2 brainstem slice preparation, contrary to our hypothesis, blocking Ih with ZD7288 caused the potentiation of inspiratory burst amplitude by muscarine to increase from 130% to 166%. Future experiments will be needed to confirm these results. Our next steps will focus on characterization of developmental changes in muscarinic modulation of XII motoneuron inspiratory burst output.

Characterizing the Action of Arginine Vasopressin at Hypoglossal Motoneurons In Neonatal Mice

Vasopressin, traditionally considered a hormone, also acts as an excitatory neurotransmitter. The effects of vasopressin have been characterized in brainstem motoneurons, including hypoglossal motoneurons (XII MNs). The XII motor nucleus receives inspiratory rhythm from the preBötzinger Complex where it is relayed to the muscles of the tongue via the XII nerve. With some variation across postnatal maturation, the vasopressin V1a receptor gene is expressed from birth to adulthood in the XII motor nucleus, and is enriched in the XII nucleus in adults compared to other cranial motoneuron populations. Previous research demonstrated that vasopressin, acting via V1a receptors on preBötzinger Complex, can stimulate breathing in adult rats by increasing breathing frequency as well as diaphragm and genioglossus (primary tongue protruder) muscle activity. Whether vasopressin can also act directly at XII MNs to potentiate inspiratory bursting has not been well characterized. Thus, the aim of this research was to characterize vasopressin effects on XII MN inspiratory bursting behavior. We generated rhythmic, transverse medullary slices (600μM) from CD1 mice of either sex (postnatal days 0–5), and recorded inspiratory activity from XII nerve rootlets using a suction electrode. We then bath applied arginine vasopressin (0.01μM and 0.1μM, AVP, V1a receptor agonist), or locally injected AVP (0.1μM and 1.0μM). In doing so, we were able to assess the effects of arginine vasopressin on XII inspiratory burst amplitude. Bath applied AVP increased inspiratory burst amplitude by 43.4%±12.5% (n=9) when applied at 0.01μM, and 56.4%±18.1% at 0.1μM. When locally applied (n=7), AVP at 0.1μM increased inspiratory burst amplitude 38.8%±6.2%, and AVP at 1.0μM caused a 26.5%±6.3% increase in inspiratory burst amplitude with a notable increase in tonic activity. In a separate set of experiments (n=8) to test whether AVP acts via V1a receptors, local AVP increased inspiratory burst amplitude by 40.2%±9.3%. Local pre‐application of the V1a receptor antagonist ((d(CH2)51Tyr(Me)2Arg8) Vasopressin, 500nM) for 60 s attenuated the response to AVP (6.7%±14.7% decrease in inspiratory burst amplitude), but the AVP response was recovered after 15 minutes with an increase in XII inspiratory burst amplitude of 22.3%±4.1%. Overall, our results support that AVP can increase XII inspiratory burst amplitude via V1a receptor activation. Further research will need to determine whether AVP plays a role in maintaining airway patency in vivo.Support or Funding InformationKenneth A. Suarez Research Fellowship, Midwestern University

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.

GABA and glycine neurons from the ventral medullary region inhibit hypoglossal motoneurons.

Obstructive sleep apnea (OSA) is a common disorder characterized by repetitive sleep-related losses of upper airway patency that occur most frequently during rapid eye movement (REM) sleep. Hypoglossal motoneurons play a key role in regulating upper airway muscle tone and patency during sleep. REM sleep activates GABA and glycine neurons in the ventral medulla (VM) to induce cortical desynchronization and skeletal muscle atonia during REM sleep; however, the role of this brain region in modulating hypoglossal motor activity is unknown. We combined optogenetic and chemogenetic approaches with in-vitro and in-vivo electrophysiology, respectfully, in GAD2-Cre mice of both sexes to test the hypothesis that VM GABA/glycine neurons control the activity of hypoglossal motoneurons and tongue muscles. Here, we show that there is a pathway originating from GABA/glycine neurons in the VM that monosynaptically inhibits brainstem hypoglossal motoneurons innervating both tongue protruder genioglossus (GMNs) and retractor (RMNs) muscles. Optogenetic activation of ChR2-expressing fibers induced a greater postsynaptic inhibition in RMNs than in GMNs. In-vivo chemogenetic activation of VM GABA/glycine neurons produced an inhibitory effect on tongue electromyographic (EMG) activity, decreasing both the amplitude and duration of inspiratory-related EMG bursts without any change in respiratory rate. These results indicate that activation of GABA/glycine neurons from the VM inhibits tongue muscles via a direct pathway to both GMNs and RMNs. This inhibition may play a role in REM sleep associated upper airway obstructions that occur in patients with OSA.

GIRK Channel Activation Contributes to the Muscarinic Modulation of XII Motoneuron Excitability in Neonatal Mice in Vitro

Sleep specific decreases in airway muscle tone, and particularly the genioglossus muscle of the tongue (primary tongue protruder), have been causally implicated in obstructive sleep apnea. During REM (rapid eye movement) sleep, decreased excitability of the XII motoneurons that innervate the genioglossus muscle may be due to a loss of excitatory noradrenergic modulation, or due to activation of muscarinic acetylcholine receptors that may act via G‐protein coupled inward rectifying potassium (GIRK) channels. GIRK channel activation results in membrane hyperpolarization and thus could mediate a decrease in excitability of XII motoneurons during REM sleep. Here we test the hypothesis that muscarine acts via GIRK channels to inhibit inspiratory burst amplitude of XII motoneurons in vitro. Using immunohistochemical techniques, our preliminary data indicate that XII motoneurons express GIRK1, GIRK2, and GIRK3 isoforms. Next, using rhythmically medullary slice preparations from neonatal CD1 mice (postnatal day, P0–5), we investigated the effects of GIRK channel and muscarinic acetycholine receptor activation on XII inspiratory burst characteristics including burst amplitude and burst period. Local application of muscarine (30 s, 100 mM) to the XII motor nucleus had the net effect of increasing XII inspiratory burst amplitude, although local pre‐application of the GIRK channel antagonist Tertiapin Q (TQ, 60 s, 100 nM) led to two different responses. In P0–2 mice (n = 4), muscarine increased burst amplitude to 120% of control (range 105–133%) while pre‐application of TQ followed by local application of muscarine led to a similar 115% (range 104–123%) increase in amplitude, suggesting that muscarine does not activate GIRK channels in P0–2 mice. In P3–5 mice (n = 4), however, local application of muscarine increased burst amplitude to 135% (range 122–156%) of baseline, and pre‐application of TQ to block GIRK channel activation further increased the response to muscarine to 160% (range 130–170%) of baseline amplitude. These results indicate that muscarine acts at GIRK channels to inhibit XII motoneuron excitability in P3–5 mice, but also acts at other channels to enhance XII motoneuron excitability. Preliminary data suggests this effect is specific to muscarinic activation of GIRK channels because bath application of TQ (10 nM) in P3–5 mice resulted in no change in inspiratory burst amplitude (mean: 102% of baseline, range 90–109%, after 30 min in TQ, n = 3). In contrast, bath application of the GIRK channel agonist ML297 (10 mM) resulted burst amplitude decrease to 80–88% of baseline in 2 out of 3 P3–5 neonatal mice and an increase in burst amplitude to 120% of baseline in a third prep. Burst period increased to 179% of baseline (range: 128–263%) indicating that GIRK channels are likely also present in preBötzinger Complex where inspiratory rhythm is generated. These data support the hypothesis that activation of muscarinic receptors in XII motoneurons leads to GIRK channel activation in P3–5 neonatal mice, which may be one mechanism through which muscarine mediates a decrease in XII motoneuron excitability in vivo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuropathic changes in the tongue protruder muscles in patients with snoring or obstructive sleep apnea

Both myopathic and neuropathic tongue protruder muscle changes have been demonstrated to occur in obstructive sleep apnea (OSA) patients using different methods. We tried to elucidate this dilemma using quantitative electromyographic (EMG) methods. In a group of consecutive patients with suspected OSA a full overnight polysomnography (PSG) and quantitative needle EMG of the tongue protruder, biceps brachii and vastus lateralis muscles were performed. EMG findings were compared to control subjects. Of 23 patients, 8 were classified as simple snorers and the remaining 15 as OSA patients by PSG. Motor unit potential (MUP) parameters obtained in tongue protruder muscles, but not biceps brachii or vastus lateralis muscles, were significantly larger in patients compared to controls. However, no correlation was found between tongue protruder muscle MUP parameters and patient characteristics. Our study confirmed previous findings of neuropathic changes in the tongue protruder muscles of OSA patients, and extended these to simple snorers. Changes were limited to the upper airway muscle, and could not been explained by the severity or duration of the sleep breathing disorder. The possible role of denervation injury caused by the upper airway vibrations should be tested in the future by appropriately designed studies.

How to dip nectar: optimal time apportionment in natural viscous fluid transport

The mouthparts of some animals are highly evolved fluid transporters. Most honeybees dip viscous nectar in a cyclic fashion by using protrusible tongues with active hairs that can erect rhythmically. The glossal hairs flatten when the tongue extends into the nectar, and then erect outwards like an umbrella to catch nectar while retracting. This paper examines the potential capability of honeybees in allocating the duration of the tongue protraction and retraction phases for the sake of energy saving. A physical model is established to analyze energy consumption induced by viscous drag, considering tongue kinematics and variation of the surface profile in different phases of tongue movements. The results indicate that the theoretically optimal time apportionment ratio at which the energy consumption is the minimum, is directly related to the square root of the tongue’s diameter ratio between the protraction and retraction phase. Through dipping observations, we validate that the duration for the protraction and retraction phases show high accordance with the theoretical prediction. These findings not only broaden the insights into honeybee’s foraging strategy but inspire the design of high-performance microfluidic pumps with dynamic surfaces to transport viscous fluid.

Relating form to function in the hummingbird feeding apparatus.

A complete understanding of the feeding structures is fundamental in order to study how animals survive. Some birds use long and protrusible tongues as the main tool to collect their central caloric source (e.g., woodpeckers and nectarivores). Hummingbirds are the oldest and most diverse clade of nectarivorous vertebrates, being a perfect subject to study tongue specializations. Their tongue functions to intraorally transport arthropods through their long bills and enables them to exploit the nectarivorous niche by collecting small amounts of liquid, therefore it is of vital importance to study its anatomy and structure at various scales. I focused on the portions of the hummingbird tongue that have been shown to be key for understanding their feeding mechanisms. I used histology, transmission and scanning electron microscopy, microCT, and ex-vivo experiments in order to advance the comprehension of the morphology and functioning of the hummingbird feeding apparatus. I found that hummingbird tongues are composed mainly of thin cornified epithelium, lack papillae, and completely fill the internal cast of the rostral oropharyngeal cavity. Understanding this puzzle-piece match between bill and tongue will be essential for the study of intraoral transport of nectar. Likewise, I found that the structural composition and tissue architecture of the tongue groove walls provide the rostral portion of the tongue with elastic properties that are central to the study of tongue-nectar interactions during the feeding process. Detailed studies on hummingbirds set the basis for comparisons with other nectar-feeding birds and contribute to comprehend the natural solutions to collecting liquids in the most efficient way possible.

Effects of aging on evoked retrusive tongue actions

ObjectiveTongue strength, timing, and coordination deficits may underlie age-related swallowing function. Retrusive tongue actions are likely important in retrograde bolus transport. However, age-related changes in retrusive tongue muscle contractile properties have not been identified in animal studies. Because previous studies employed whole hypoglossal nerve stimulation that activated both protrusive and retrusive tongue muscles, co-contraction may have masked retrusive muscle force decrements. The hypotheses of this study were: (1) retrusive tongue muscle contraction forces would be diminished and temporal characteristics prolonged in old rats when lateral nerves were selectively activated, and (2) greater muscle contractile forces with selective lateral branch stimulation would be found relative to whole hypoglossal nerve stimulation. DesignNineteen Fischer 344/Brown Norway rats (9 old, 10 young adult) underwent tongue muscle contractile property recording elicited by: (1) bilateral whole hypoglossal nerve stimulation, and (2) selective lateral branch stimulation. Twitch contraction time (CT), half-decay time, maximal twitch and tetanic forces, and a fatigue index were measured. ResultsFor whole nerve stimulation, CT was significantly longer in the old group. No significant age group differences were found with selective lateral nerve stimulation. Significantly reduced twitch forces (old group only), increased tetanic forces and significantly less fatigue were found with selective lateral nerve stimulation than with whole hypoglossal stimulation. ConclusionsRetrusive tongue forces are not impaired in old rats. Deficits observed in swallowing with aging may be due to other factors such as inadequate bolus propulsive forces, mediated by protrusive tongue muscles, or timing/coordination of muscle actions.

Maximum protrusive tongue force in healthy young adults

In clinical speech-language pathology practice, tongue force is usually evaluated qualitatively. Perception and practical experience are used to classify this force. The Biomechanical Engineering Group from the Federal University of Minas Gerais developed an instrument to quantify tongue force. The purposes of this study were to quantify maximum tongue protrusion force in Brazilian subjects with normal tongue strength and to compare force values between gender groups. In total, 105 subjects, 43 men and 62 women, aged from 18 to 29 years, with normal tongue strength according to qualitative evaluation, underwent quantitative evaluation by using the instrument. The mean of the maximum tongue force values of all participants was 17.58±7.95 N. There were significant differences in the median values for maximum tongue forces between the genders, with higher values observed for men. In intersubject comparisons, high variation coefficients were evident due to the variability among individuals. However, the study suggested that the instrument could be an interesting tool for intrasubject comparisons, especially during the follow-up.

Protrusive tongue strength in people with severely weak tongues

The purpose of the study was to quantify tongue protrusion force and compare its characteristics between participants with severely weak tongues and those with normal lingual strength. The sample consisted of 11 participants with severe lingual strength deficits and 11 age- and sex-matched participants with normal lingual strength. Tongue force was evaluated quantitatively using the Forling instrument, and the average force, maximum force, average force application rate, and area under the graphic curve were analysed. These parameters were compared between the groups. In the participants with severely weak tongues, the average and the maximum forces in N (Newton) were 2.03 ± 1.17 and 3.56 ± 1.77, respectively. The average force application rate in N/s (Newton per second) was 1.25 and the area under the graphic curve in Ns (Newton times second) was 18.6. The values of the participants with normal lingual strength were, respectively, 13.27 ± 6.15 N, 18.91 ± 7.95 N, 10.46 N/s, and 108.08 Ns. All parameters analysed differed significantly between the groups. The data collected could aid speech-language pathologists in diagnosing problems related to tongue force.