Regional associations between inspiratory tongue dilatory movement and genioglossus activity during wakefulness in people with obstructive sleep apnoea.

Inspiratory tongue dilatory movement is believed to be mediated via changes in neural drive to genioglossus. However, this has not been studied during quiet breathing in humans. Therefore, this study investigated this relationship and its potential role in obstructive sleep apnoea (OSA). During awake supine quiet nasal breathing, inspiratory tongue dilatory movement, quantified with tagged magnetic resonance imaging, and inspiratory phasic genioglossus EMG normalised to maximum EMG were measured in nine controls [apnoea-hypopnea index (AHI) ≤5 events/h] and 37 people with untreated OSA (AHI >5 events/h). Measurements were obtained for 156 neuromuscular compartments (85%). Analysis was adjusted for nadir epiglottic pressure during inspiration. Only for 106 compartments (68%) was a larger anterior (dilatory) movement associated with a higher phasic EMG [mixed linear regression, beta = 0.089, 95% CI [0.000, 0.178], t(99) = 1.995, P = 0.049, hereafter EMG↗/mvt↗]. For the remaining 50 (32%) compartments, a larger dilatory movement was associated with a lower phasic EMG [mixed linear regression, beta = -0.123, 95% CI [-0.224, -0.022], t(43) = -2.458, P = 0.018, hereafter EMG↘/mvt↗]. OSA participants had a higher odds of having at least one decoupled EMG↘/mvt↗ compartment (binary logistic regression, odds ratio [95% CI]: 7.53 [1.19, 47.47] (P = 0.032). Dilatory tongue movement was minimal (>1 mm) in nearly all participants with only EMG↗/mvt↗ compartments (86%, 18/21). These results demonstrate that upper airway dilatory mechanics cannot be predicted from genioglossus EMG, particularly in people with OSA. Tongue movement associated with minimal genioglossus activity suggests co-activation of other airway dilator muscles. KEY POINTS: Inspiratory tongue movement is thought to be mediated through changes in genioglossus activity. However, it is unknown if this relationship is altered by obstructive sleep apnoea (OSA). During awake supine quiet nasal breathing, inspiratory tongue movement, quantified with tagged magnetic resonance imaging (MRI), and inspiratory phasic genioglossus EMG normalised to maximum EMG were measured in four tongue compartments of people with and without OSA. Larger tongue anterior (dilatory) movement was associated with higher phasic genioglossus EMG for 68% of compartments. OSA participants had an ∼7-times higher odds of having at least one compartment for which a larger anterior tongue movement was not associated with a higher phasic EMG than controls. Therefore, higher genioglossus phasic EMG does not consistently translate into tongue dilatory movement, particularly in people with OSA. Large dilatory tongue movements can occur despite minimal genioglossus inspiratory activity, suggesting co-activation of other pharyngeal muscles.

Descending pathways from the superior colliculus mediating autonomic and respiratory effects associated with orienting behaviour.

The ability to discriminate competing external stimuli and initiate contextually appropriate behaviours is a key brain function. Neurons in the deep superior colliculus (dSC) integrate multisensory inputs and activate descending projections to premotor pathways responsible for orienting, attention and defence, behaviours which involve adjustments to respiratory and cardiovascular parameters. However, the neural pathways that subserve the physiological components of orienting are poorly understood. We report that orienting responses to optogenetic dSC stimulation are accompanied by short-latency autonomic, respiratory and electroencephalographic effects in awake rats, closely mimicking those evoked by naturalistic alerting stimuli. Physiological responses were not accompanied by detectable aversion or fear, and persisted under urethane anaesthesia, indicating independence from emotional stress. Anterograde and trans-synaptic viral tracing identified a monosynaptic pathway that links the dSC to spinally projecting neurons in the medullary gigantocellular reticular nucleus (GiA), a key hub for the coordination of orienting and locomotor behaviours. In urethane-anaesthetized animals, sympathoexcitatory and cardiovascular, but not respiratory, responses to dSC stimulation were replicated by optogenetic stimulation of the dSC-GiA terminals, suggesting a likely role for this pathway in mediating the autonomic components of dSC-mediated responses. Similarly, extracellular recordings from putative GiA sympathetic premotor neurons confirmed short-latency excitatory inputs from the dSC. This pathway represents a likely substrate for autonomic components of orienting responses that are mediated by dSC neurons and suggests a mechanism through which physiological and motor components of orienting behaviours may be integrated without the involvement of higher centres that mediate affective components of defensive responses. KEY POINTS: Neurons in the deep superior colliculus (dSC) integrate multimodal sensory signals to elicit context-dependent innate behaviours that are accompanied by stereotypical cardiovascular and respiratory activities. The pathways responsible for mediating the physiological components of colliculus-mediated orienting behaviours are unknown. We show that optogenetic dSC stimulation evokes transient orienting, respiratory and autonomic effects in awake rats which persist under urethane anaesthesia. Anterograde tracing from the dSC identified projections to spinally projecting neurons in the medullary gigantocellular reticular nucleus (GiA). Stimulation of this pathway recapitulated autonomic effects evoked by stimulation of dSC neurons. Electrophysiological recordings from putative GiA sympathetic premotor neurons confirmed short latency excitatory input from dSC neurons. This disynaptic dSC-GiA-spinal sympathoexcitatory pathway may underlie autonomic adjustments to salient environmental cues independent of input from higher centres.

Upregulated Angiotensin Ia Receptors in the Hypothalamic Paraventricular Nucleus Sensitize Neuroendocrine Vasopressin Release and Blood Pressure in a Rodent Model of Polycystic Kidney Disease.

INTRODUCTION: Angiotensin (Ang) II signalling in the hypothalamic paraventricular nucleus (PVN) via Ang type-1a receptors (AT1R) regulates vasopressin release and sympathetic nerve activity - two effectors of blood pressure regulation. We determined the cellular expression and function of AT1R in the PVN of a rodent model of polycystic kidney disease (PKD), the Lewis polycystic kidney (LPK) rat, to evaluate its contribution to blood pressure regulation and augmented vasopressin release in PKD. METHODS: PVN AT1R gene expression was quantified with fluorescent in situ hybridization in LPK and control rats. PVN AT1R function was assessed with pharmacology under urethane anaesthesia in LPK and control rats instrumented to record arterial pressure and sympathetic nerve activity. RESULTS: AT1R gene expression was upregulated in the PVN, particularly in corticotrophin-releasing hormone neurons, of LPK versus control rats. PVN microinjection of Ang II produced larger increases in systolic blood pressure in LPK versus control rats (36 ± 5 vs. 17 ± 2 mm Hg; p < 0.01). Unexpectedly, Ang II produced regionally heterogeneous sympathoinhibition (renal: -33%; splanchnic: -12%; lumbar: no change) in LPK and no change in controls. PVN pre-treatment with losartan, a competitive AT1R antagonist, blocked the Ang II-mediated renal sympathoinhibition and attenuated the pressor response observed in LPK rats. The Ang II pressor effect was also blocked by systemic OPC-21268, a competitive V1A receptor antagonist, but unaffected by hexamethonium, a sympathetic ganglionic blocker. DISCUSSION/CONCLUSION: Collectively, our data suggest that upregulated AT1R expression in PVN sensitizes neuroendocrine release of vasopressin in the LPK, identifying a central mechanism for the elevated vasopressin levels present in PKD.

Regional respiratory movement of the tongue is coordinated during wakefulness and is larger in severe obstructive sleep apnoea.

KEY POINTS: Coordination of the neuromuscular compartments of the tongue is critical to maintain airway patency. Currently, little is known about the extent to which regional tongue dilatory motion is coordinated in heathy people and if this coordination is altered in people with obstructive sleep apnoea (OSA). We show that regional tongue muscle coordination in people with and without OSA during wakefulness is associated with effective airway dilatation during inspiration, using dynamic tagged magnetic resonance imaging. The maximal movement of four compartments of the tongue were correlated and occurred concurrently towards the end of inspiration. If tongue movement was observed, people with more severe OSA had larger movement and moved more compartments (up to four) to maintain airway patency, while people without OSA moved only one compartment. These results suggest that airway patency is preserved during wakefulness in people with OSA via active dilatory movement of the genioglossus. ABSTRACT: Maintaining airway patency when supine requires neural drive to the genioglossus horizontal and oblique neuromuscular compartments (superior fan-like and inferior horizontal genioglossus, regions that are innervated by different branches of the hypoglossal nerve) to be coordinated during breathing, but it is unknown if this coordination is altered in obstructive sleep apnoea (OSA). This study aimed to assess coordination of airway dilatory motion across four mid-sagittal tongue compartments during inspiration (i.e. anterior and posterior of the horizontal and oblique compartments), and compare it in controls and OSA patients. Fifty-four participants (12 women, aged 20-73 years) underwent dynamic 'tagged' magnetic resonance imaging during wakefulness. Ten participants had no OSA [apnoea hypopnoea index (AHI) < 5 events h-1 ], 14 had mild OSA (5 < AHI ≤ 15 events h-1 ), 12 had moderate OSA (15 < AHI ≤ 30 events h-1 ) and 18 had severe OSA (AHI > 30 events h-1 ). A higher AHI was associated with a greater anterior movement of the anterior and posterior horizontal compartments (Spearman, r = -0.32, P = 0.02 for both), but not in the oblique compartments. If movement was observed, higher OSA severity was associated with an anterior movement of a greater number of compartments. Controls only moved the posterior horizontal compartment while the anterior horizontal compartment also moved in OSA participants. Oblique compartments moved only in people with severe OSA. The maximal anterior inspiratory movement of the four compartments was highly correlated (Spearman, P < 0.001) and occurred concurrently. The posterior horizontal compartment had the greatest anterior motion. These results suggest that airway patency is preserved during wakefulness in people with OSA via active dilatory movement of the genioglossus.

Polysialic Acid Regulates Sympathetic Outflow by Facilitating Information Transfer within the Nucleus of the Solitary Tract.

Expression of the large extracellular glycan, polysialic acid (polySia), is restricted in the adult, to brain regions exhibiting high levels of plasticity or remodeling, including the hippocampus, prefrontal cortex, and the nucleus of the solitary tract (NTS). The NTS, located in the dorsal brainstem, receives constant viscerosensory afferent traffic as well as input from central regions controlling sympathetic nerve activity, respiration, gastrointestinal functions, hormonal release, and behavior. Our aims were to determine the ultrastructural location of polySia in the NTS and the functional effects of enzymatic removal of polySia, both in vitro and in vivo polySia immunoreactivity was found throughout the adult rat NTS. Electron microscopy demonstrated polySia at sites that influence neurotransmission: the extracellular space, fine astrocytic processes, and neuronal terminals. Removing polySia from the NTS had functional consequences. Whole-cell electrophysiological recordings revealed altered intrinsic membrane properties, enhancing voltage-gated K+ currents and increasing intracellular Ca2+ Viscerosensory afferent processing was also disrupted, dampening low-frequency excitatory input and potentiating high-frequency sustained currents at second-order neurons. Removal of polySia in the NTS of anesthetized rats increased sympathetic nerve activity, whereas functionally related enzymes that do not alter polySia expression had little effect. These data indicate that polySia is required for the normal transmission of information through the NTS and that changes in its expression alter sympathetic outflow. polySia is abundant in multiple but discrete brain regions, including sensory nuclei, in both the adult rat and human, where it may regulate neuronal function by mechanisms identified here.SIGNIFICANCE STATEMENT All cells are coated in glycans (sugars) existing predominantly as glycolipids, proteoglycans, or glycoproteins formed by the most complex form of posttranslational modification, glycosylation. How these glycans influence brain function is only now beginning to be elucidated. The adult nucleus of the solitary tract has abundant polysialic acid (polySia) and is a major site of integration, receiving viscerosensory information which controls critical homeostatic functions. Our data reveal that polySia is a determinant of neuronal behavior and excitatory transmission in the nucleus of the solitary tract, regulating sympathetic nerve activity. polySia is abundantly expressed at distinct brain sites in adult, including major sensory nuclei, suggesting that sensory transmission may also be influenced via mechanisms described here. These findings hint at the importance of elucidating how other glycans influence neural function.

Hypoxia silences retrotrapezoid nucleus respiratory chemoreceptors via alkalosis.

In conscious mammals, hypoxia or hypercapnia stimulates breathing while theoretically exerting opposite effects on central respiratory chemoreceptors (CRCs). We tested this theory by examining how hypoxia and hypercapnia change the activity of the retrotrapezoid nucleus (RTN), a putative CRC and chemoreflex integrator. Archaerhodopsin-(Arch)-transduced RTN neurons were reversibly silenced by light in anesthetized rats. We bilaterally transduced RTN and nearby C1 neurons with Arch (PRSx8-ArchT-EYFP-LVV) and measured the cardiorespiratory consequences of Arch activation (10 s) in conscious rats during normoxia, hypoxia, or hyperoxia. RTN photoinhibition reduced breathing equally during non-REM sleep and quiet wake. Compared with normoxia, the breathing frequency reduction (Δf(R)) was larger in hyperoxia (65% FiO2), smaller in 15% FiO2, and absent in 12% FiO2. Tidal volume changes (ΔV(T)) followed the same trend. The effect of hypoxia on Δf(R) was not arousal-dependent but was reversed by reacidifying the blood (acetazolamide; 3% FiCO2). Δf(R) was highly correlated with arterial pH up to arterial pH (pHa) 7.5 with no frequency inhibition occurring above pHa 7.53. Blood pressure was minimally reduced suggesting that C1 neurons were very modestly inhibited. In conclusion, RTN neurons regulate eupneic breathing about equally during both sleep and wake. RTN neurons are the first putative CRCs demonstrably silenced by hypocapnic hypoxia in conscious mammals. RTN neurons are silent above pHa 7.5 and increasingly active below this value. During hyperoxia, RTN activation maintains breathing despite the inactivity of the carotid bodies. Finally, during hypocapnic hypoxia, carotid body stimulation increases breathing frequency via pathways that bypass RTN.

Proton detection and breathing regulation by the retrotrapezoid nucleus.

We discuss recent evidence which suggests that the principal central respiratory chemoreceptors are located within the retrotrapezoid nucleus (RTN) and that RTN neurons are directly sensitive to [H(+) ]. RTN neurons are glutamatergic. In vitro, their activation by [H(+) ] requires expression of a proton-activated G protein-coupled receptor (GPR4) and a proton-modulated potassium channel (TASK-2) whose transcripts are undetectable in astrocytes and the rest of the lower brainstem respiratory network. The pH response of RTN neurons is modulated by surrounding astrocytes but genetic deletion of RTN neurons or deletion of both GPR4 and TASK-2 virtually eliminates the central respiratory chemoreflex. Thus, although this reflex is regulated by innumerable brain pathways, it seems to operate predominantly by modulating the discharge rate of RTN neurons, and the activation of RTN neurons by hypercapnia may ultimately derive from their intrinsic pH sensitivity. RTN neurons increase lung ventilation by stimulating multiple aspects of breathing simultaneously. They stimulate breathing about equally during quiet wake and non-rapid eye movement (REM) sleep, and to a lesser degree during REM sleep. The activity of RTN neurons is regulated by inhibitory feedback and by excitatory inputs, notably from the carotid bodies. The latter input operates during normo- or hypercapnia but fails to activate RTN neurons under hypocapnic conditions. RTN inhibition probably limits the degree of hyperventilation produced by hypocapnic hypoxia. RTN neurons are also activated by inputs from serotonergic neurons and hypothalamic neurons. The absence of RTN neurons probably underlies the sleep apnoea and lack of chemoreflex that characterize congenital central hypoventilation syndrome.

Tonically Active cAMP-Dependent Signaling in the Ventrolateral Medulla Regulates Sympathetic and Cardiac Vagal Outflows.

The ventrolateral medulla contains presympathetic and vagal preganglionic neurons that control vasomotor and cardiac vagal tone, respectively. G protein-coupled receptors influence the activity of these neurons. Gα s activates adenylyl cyclases, which drive cyclic adenosine monophosphate (cAMP)-dependent targets: protein kinase A (PKA), the exchange protein activated by cAMP (EPAC), and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The aim was to determine the cardiovascular effects of activating and inhibiting these targets at presympathetic and cardiac vagal preganglionic neurons. Urethane-anesthetized rats were instrumented to measure splanchnic sympathetic nerve activity (sSNA), arterial pressure (AP), heart rate (HR), as well as baroreceptor and somatosympathetic reflex function, or were spinally transected and instrumented to measure HR, AP, and cardiac baroreflex function. All drugs were injected bilaterally. In the rostral ventrolateral medulla (RVLM), Sp-cAMPs and 8-Br-cAMP, which activate PKA, as well as 8-pCPT, which activates EPAC, increased sSNA, AP, and HR. Sp-cAMPs also facilitated the reflexes tested. Sp-cAMPs also increased cardiac vagal drive and facilitated cardiac baroreflex sensitivity. Blockade of PKA, using Rp-cAMPs or H-89 in the RVLM, increased sSNA, AP, and HR and increased HR when cardiac vagal preganglionic neurons were targeted. Brefeldin A, which inhibits EPAC, and ZD7288, which inhibits HCN channels, each alone had no effect. Cumulative, sequential blockade of all three inhibitors resulted in sympathoinhibition. The major findings indicate that Gα s-linked receptors in the ventral medulla can be recruited to drive both sympathetic and parasympathetic outflows and that tonically active PKA-dependent signaling contributes to the maintenance of both sympathetic vasomotor and cardiac vagal tone.

State-dependent control of breathing by the retrotrapezoid nucleus.

KEY POINTS: This study explores the state dependence of the hypercapnic ventilatory reflex (HCVR). We simulated an instantaneous increase or decrease of central chemoreceptor activity by activating or inhibiting the retrotrapezoid nucleus (RTN) by optogenetics in conscious rats. During quiet wake or non-REM sleep, hypercapnia increased both breathing frequency (fR ) and tidal volume (VT ) whereas, in REM sleep, hypercapnia increased VT exclusively. Optogenetic inhibition of RTN reduced VT in all sleep-wake states, but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Phasic RTN stimulation produced active expiration and reduced early expiratory airflow (i.e. increased upper airway resistance) only during wake. We conclude that the HCVR is highly state-dependent. The HCVR is reduced during REM sleep because fR is no longer under chemoreceptor control and thus could explain why central sleep apnoea is less frequent in REM sleep. ABSTRACT: Breathing has different characteristics during quiet wake, non-REM or REM sleep, including variable dependence on PCO2. We investigated whether the retrotrapezoid nucleus (RTN), a proton-sensitive structure that mediates a large portion of the hypercapnic ventilatory reflex, regulates breathing differently during sleep vs. wake. Electroencephalogram, neck electromyogram, blood pressure, respiratory frequency (fR ) and tidal volume (VT ) were recorded in 28 conscious adult male Sprague-Dawley rats. Optogenetic stimulation of RTN with channelrhodopsin-2, or inhibition with archaerhodopsin, simulated an instantaneous increase or decrease of central chemoreceptor activity. Both opsins were delivered with PRSX8-promoter-containing lentiviral vectors. RTN and catecholaminergic neurons were transduced. During quiet wake or non-REM sleep, hypercapnia (3 or 6% FI,CO2 ) increased both fR and VT whereas, in REM sleep, hypercapnia increased VT exclusively. RTN inhibition always reduced VT but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Blood pressure was unaffected by either stimulation or inhibition. Except in REM sleep, phasic RTN stimulation entrained and shortened the breathing cycle by selectively shortening the post-inspiratory phase. Phasic stimulation also produced active expiration and reduced early expiratory airflow but only during wake. VT is always regulated by RTN and CO2 but fR is regulated by CO2 and RTN only when the brainstem pattern generator is in autorhythmic mode (anaesthesia, non-REM sleep, quiet wake). The reduced contribution of RTN to breathing during REM sleep could explain why certain central apnoeas are less frequent during this sleep stage.

Optogenetic stimulation of adrenergic C1 neurons causes sleep state-dependent cardiorespiratory stimulation and arousal with sighs in rats.

RATIONALE: The rostral ventrolateral medulla (RVLM) contains central respiratory chemoreceptors (retrotrapezoid nucleus, RTN) and the sympathoexcitatory, hypoxia-responsive C1 neurons. Simultaneous optogenetic stimulation of these neurons produces vigorous cardiorespiratory stimulation, sighing, and arousal from non-REM sleep. OBJECTIVES: To identify the effects that result from selectively stimulating C1 cells. METHODS: A Cre-dependent vector expressing channelrhodopsin 2 (ChR2) fused with enhanced yellow fluorescent protein or mCherry was injected into the RVLM of tyrosine hydroxylase (TH)-Cre rats. The response of ChR2-transduced neurons to light was examined in anesthetized rats. ChR2-transduced C1 neurons were photoactivated in conscious rats while EEG, neck muscle EMG, blood pressure (BP), and breathing were recorded. MEASUREMENTS AND MAIN RESULTS: Most ChR2-expressing neurons (95%) contained C1 neuron markers and innervated the spinal cord. RTN neurons were not transduced. While the rats were under anesthesia, the C1 cells were faithfully activated by each light pulse up to 40 Hz. During quiet resting and non-REM sleep, C1 cell stimulation (20 s, 2-20 Hz) increased BP and respiratory frequency and produced sighs and arousal from non-REM sleep. Arousal was frequency-dependent (85% probability at 20 Hz). Stimulation during REM sleep increased BP, but had no effect on EEG or breathing. C1 cell-mediated breathing stimulation was occluded by hypoxia (12% FIO2), but was unchanged by 6% FiCO2. CONCLUSIONS: C1 cell stimulation reproduces most effects of acute hypoxia, specifically cardiorespiratory stimulation, sighs, and arousal. C1 cell activation likely contributes to the sleep disruption and adverse autonomic consequences of sleep apnea. During hypoxia (awake) or REM sleep, C1 cell stimulation increases BP but no longer stimulates breathing.

C1 neurons: the body's EMTs.

The C1 neurons reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM, IVLM). They use glutamate as a fast transmitter and synthesize catecholamines plus various neuropeptides. These neurons regulate the hypothalamic pituitary axis via direct projections to the paraventricular nucleus and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. The presympathetic C1 cells, located in the RVLM, are probably organized in a roughly viscerotopic manner and most of them regulate the circulation. C1 cells are variously activated by hypoglycemia, infection or inflammation, hypoxia, nociception, and hypotension and contribute to most glucoprivic responses. C1 cells also stimulate breathing and activate brain stem noradrenergic neurons including the locus coeruleus. Based on the various effects attributed to the C1 cells, their axonal projections and what is currently known of their synaptic inputs, subsets of C1 cells appear to be differentially recruited by pain, hypoxia, infection/inflammation, hemorrhage, and hypoglycemia to produce a repertoire of stereotyped autonomic, metabolic, and neuroendocrine responses that help the organism survive physical injury and its associated cohort of acute infection, hypoxia, hypotension, and blood loss. C1 cells may also contribute to glucose and cardiovascular homeostasis in the absence of such physical stresses, and C1 cell hyperactivity may contribute to the increase in sympathetic nerve activity associated with diseases such as hypertension.

Direct optogenetic activation of upper airway muscles in an acute model of upper airway hypotonia mimicking sleep onset.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA), where the upper airway collapses repeatedly during sleep due to inadequate dilator muscle tone, is challenging to treat as current therapies are poorly tolerated or have variable and unpredictable efficacy. We propose a novel, optogenetics-based therapy, that stimulates upper airway dilator muscle contractions in response to light. To determine the feasibility of a novel optogenetics-based OSA therapy, we developed a rodent model of human sleep-related upper airway muscle atonia. Using this model, we evaluated intralingual delivery of candidate optogenetic constructs, notably a muscle-targeted approach that will likely have a favorable safety profile. METHODS: rAAV serotype 9 viral vectors expressing a channelrhodopsin-2 variant, driven by a muscle-specific or nonspecific promoter were injected into rat tongues to compare strength and specificity of opsin expression. Light-evoked electromyographic responses were recorded in an acute, rodent model of OSA. Airway dilation was captured with ultrasound. RESULTS: The muscle-specific promoter produced sufficient opsin expression for light stimulation to restore and/or enhance electromyographic signals (linear mixed model, F = 140.0, p < 0.001) and induce visible tongue contraction and airway dilation. The muscle-specific promoter induced stronger (RM-ANOVA, F(1,8) = 10.0, p = 0.013) and more specific opsin expression than the nonspecific promoter in an otherwise equivalent construct. Viral DNA and RNA were robust in the tongue, but low or absent in all other tissues. CONCLUSIONS: Significant functional responses to direct optogenetic muscle activation were achieved following muscle-specific promoter-driven rAAV-mediated transduction, providing proof-of-concept for an optogenetic therapy for patients with inadequate dilator muscle activity during sleep.

Do catecholaminergic TrkC DRG neurons represent a class of cardiovascular enteroceptor?

In a recent issue of Cell Reports, Morelli et al. (2021) identify a subpopulation of mechanosensitive peripheral sensory neurons that coexpress tyrosine hydroxylase (TH) and tropomyosin receptor kinase C (TrkC) and innervate cutaneous arterioles. They show that activation of TrkC sensory neurons causes cutaneous vasoconstriction and, most remarkably, that their lesion is associated with sudden death of an undetermined cause, preceded by a progressive drop in blood pressure, and conclude that TrkC+ TH+ neurons represent a baroreceptor class of homeostatic enteroceptor. This represents a radical departure from current consensus models for the central control of blood pressure. Here, we offer an alternative perspective on their findings and suggest priorities for further investigation. This Matters Arising paper is in response to Morelli et al. (2021), published in Cell Reports. See also the response by Heppenstall et al. (2022), published in this issue.

Regional genioglossus reflex responses to negative pressure pulses in people with obstructive sleep apnea.

Tongue and upper airway dilator muscle movement patterns during quiet breathing vary in people with obstructive sleep apnea (OSA). Many patients have inadequate or counterproductive responses to inspiratory negative airway pressure that likely contributes to their OSA. This may be due, at least in part, to inadequate or nonhomogeneous reflex drive to different regions of the largest upper airway dilator, genioglossus. To investigate potential regional heterogeneity of genioglossus reflex responses in OSA, brief suction pulses were applied via a nasal breathing mask and an electromyogram (EMG) was recorded in four regions (anterior oblique, anterior horizontal, posterior oblique, and posterior horizontal) using intramuscular fine wire electrodes in 15 people with OSA. Genioglossus short-latency reflex excitation amplitude had regional heterogeneity (horizontal vs. oblique regions) when expressed in absolute units but homogeneity when normalized as a percentage of the immediate (100 ms) prestimulus EMG. Regional variability in reflex morphology (excitation and inhibition) was present in one-third of the participants. The minimum cross-sectional area (CSA) of the pharyngeal airway was quantified using MRI and may be related to the amplitude of the short-latency reflex response to negative pressure as we found that people with a smaller CSA tended to have a greater reflex amplitude (e.g., horizontal region r2 = 0.41, P = 0.01). These findings highlight the complexity of genioglossus reflex control, the potential for regional heterogeneity, and the functional importance of upper airway anatomy in mediating genioglossus reflex responses to rapid changes in negative pressure in OSA.NEW & NOTEWORTHY Our findings indicate that 30% of participants had regional heterogeneity in reflex morphology (excitation/inhibition) to brief pulses of negative upper-airway pressure across anterior oblique, anterior horizontal, posterior oblique, and posterior horizontal regions of the genioglossus muscle. Reflex excitation amplitude was proportional to prestimulus drive, with increased activation in oblique compared with horizontal regions of the posterior tongue. People with narrower upper-airway anatomy tended to have increased genioglossus reflex amplitude to negative pressure pulses during wakefulness.

The relationship between mandibular advancement, tongue movement, and treatment outcome in obstructive sleep apnea.

STUDY OBJECTIVES: To characterize how mandibular advancement enlarges the upper airway via posterior tongue advancement in people with obstructive sleep apnea (OSA) and whether this is associated with mandibular advancement splint (MAS) treatment outcome. METHODS: One-hundred and one untreated people with OSA underwent a 3T magnetic resonance (MRI) scan. Dynamic mid-sagittal posterior tongue and mandible movements during passive jaw advancement were measured with tagged MRI. Upper airway cross-sectional areas were measured with the mandible in a neutral position and advanced to 70% of maximum advancement. Treatment outcome was determined after a minimum of 9 weeks of therapy. RESULTS: Seventy-one participants completed the study: 33 were responders (AHI<5 or AHI≤10 events/hr with >50% AHI reduction), 11 were partial responders (>50% AHI reduction but AHI>10 events/hr), and 27 nonresponders (AHI reduction<50% and AHI≥10 events/hr). Responders had the greatest naso- and oropharyngeal tongue anterior movement (0.40 ± 0.08 and 0.47 ± 0.13 mm, respectively) and oropharyngeal cross-sectional area enlargement (6.41 ± 2.12%) per millimeter of mandibular advancement. A multivariate model that included tongue movement and percentage of airway enlargement per millimeter of mandibular advancement along with baseline AHI correctly classified 69.2% (5-fold cross-validated 62.5%, n = 39) of participants in response categories when the jaw was advanced in the range that would usually be regarded as sufficient for clinical efficacy (> 4 mm). In comparison, a model using only baseline AHI correctly classified 50.0% of patients (5-fold cross-validated 52.5%, n = 40). CONCLUSIONS: Tongue advancement and upper airway enlargement with mandibular advancement in conjunction with baseline AHI improve treatment response categorization to a satisfactory level (69.2%, 5-fold cross-validated 62.5%).

Task-dependent neural control of regions within human genioglossus.

Anatomical and imaging evidence suggests neural control of oblique and horizontal compartments of the genioglossus differs. However, neurophysiological evidence for differential control remains elusive. This study aimed to determine whether there are differences in neural drive to the oblique and horizontal regions of the genioglossus during swallowing and tongue protrusion. Adult participants (n = 63; 48 M) were recruited from a sleep clinic; 41 had obstructive sleep apnea (OSA: 34 M, 8 F). Electromyographic (EMG) was recorded at rest (awake, supine) using four intramuscular fine-wire electrodes inserted percutaneously into the anterior oblique, posterior oblique, anterior horizontal, and posterior horizontal genioglossus. Epiglottic pressure and nasal airflow were also measured. During swallowing, two distinct EMG patterns were observed - a monophasic response (single EMG peak) and a biphasic response (2 bursts of EMG). Peak EMG and timing of the peak relative to epiglottic pressure were significantly different between patterns (linear mixed models, P < 0.001). Monophasic activation was more likely in the horizontal than oblique region during swallowing (OR = 6.83, CI = 3.46-13.53, P < 0.001). In contrast, during tongue protrusion, activation patterns and EMG magnitude were not different between regions. There were no systematic differences in EMG patterns during swallowing or tongue protrusion between OSA and non-OSA groups. These findings provide evidence for functional differences in the motoneuronal output to the oblique and horizontal compartments, enabling differential task-specific drive. Given this, it is important to identify the compartment from which EMG is acquired. We propose that the EMG patterns during swallowing may be used to identify the compartment where a recording electrode is located.NEW & NOTEWORTHY During swallowing, we observed two distinct, stereotyped muscle activation patterns that define the horizontal (monophasic, maximal EMG) and oblique (biphasic, submaximal EMG) neuromuscular compartments of genioglossus. In contrast, volitional tongue protrusions produced uniform activation across compartments. This provides evidence for task-dependent, functionally discrete neuromuscular control of the oblique and horizontal compartments of genioglossus. The magnitude and temporal patterning of genioglossus EMG during swallowing may help guide electrode placement in tongue EMG studies.

Patterning of somatosympathetic reflexes reveals nonuniform organization of presympathetic drive from C1 and non-C1 RVLM neurons.

To determine the organization of presympathetic vasomotor drive by phenotypic populations of rostral ventrolateral medulla (RVLM) neurons, we examined the somatosympathetic reflex (SSR) evoked in four sympathetic nerves together with selective lesions of RVLM presympathetic neurons. Urethane-anesthetized (1.3 g/kg ip), paralyzed, vagotomized and artificially ventilated Sprague-Dawley rats (n = 41) were used. First, we determined the afferent inputs activated by sciatic nerve (SN) stimulation at graded stimulus intensities (50 sweeps at 0.5-1 Hz, 1-80 V). Second, we recorded sympathetic nerve responses (cervical, renal, splanchnic, and lumbar) to intensities of SN stimulation that activated A-fiber afferents (low) or both A- and C-fiber afferents (high). Third, with low-intensity SN stimulation, we examined the cervical SSR following RVLM microinjection of somatostatin, and we determined the splanchnic SSR in rats in which presympathetic C1 neurons were lesioned following intraspinal injections of anti-dopamine-ß-hydroxylase-saporin (anti-DßH-SAP). Low-intensity SN stimulation activated A-fiber afferents and evoked biphasic responses in the renal, splanchnic, and lumbar nerves and a single peak in the cervical nerve. Depletion of presympathetic C1 neurons (59 ± 4% tyrosine hydroxylase immunoreactivity profiles lesioned) eliminated peak 2 of the splanchnic SSR and attenuated peak 1, suggesting that only RVLM neurons with fast axonal conduction were spared. RVLM injections of somatostatin abolished the single early peak of cervical SSR confirming that RVLM neurons with fast axonal conduction were inhibited by somatostatin. It is concluded that unmyelinated RVLM presympathetic neurons, presumed to be all C1, innervate splanchnic, renal, and lumbar but not cervical sympathetic outflows, whereas myelinated C1 and non-C1 RVLM neurons innervate all sympathetic outflows examined. These findings suggest that multiple levels of neural control of vasomotor tone exist; myelinated populations may set baseline tone, while unmyelinated neurons may be recruited to provide actions at specific vascular beds in response to distinct stressors.

Augmented Respiratory-Sympathetic Coupling and Hemodynamic Response to Acute Mild Hypoxia in Female Rodents With Chronic Kidney Disease.

Carotid body feedback and hypoxia may serve to enhance respiratory-sympathetic nerve coupling (respSNA) and act as a driver of increased blood pressure. Using the Lewis polycystic kidney (LPK) rat model of chronic kidney disease, we examined respSNA in adult female rodents with CKD and their response to acute hypoxia or hypercapnia compared to Lewis control animals. Under urethane anesthesia, phrenic nerve activity, splanchnic sympathetic nerve activity (sSNA), and renal sympathetic nerve activity (rSNA) were recorded under baseline conditions and during mild hypoxic or hypercapnic challenges. At baseline, tonic SNA and blood pressure were greater in female LPK rats versus Lewis rats (all P < 0.05) and respSNA was at least two-fold larger [area under the curve (AUC), sSNA: 7.8 ± 1.1 vs. 3.4 ± 0.7 µV s, rSNA: 11.5 ± 3 vs. 4.8 ± 0.7 µV s, LPK vs. Lewis, both P < 0.05]. Mild hypoxia produced a larger pressure response in LPK [Δ mean arterial pressure (MAP) 30 ± 6 vs. 12 ± 6 mmHg] and augmented respSNA (ΔAUC, sSNA: 8.9 ± 3.4 vs. 2 ± 0.7 µV s, rSNA: 6.1 ± 1.2 vs. 3.1 ± 0.7 µV s, LPK vs. Lewis, all P ≤ 0.05). In contrast, central chemoreceptor stimulation produced comparable changes in blood pressure and respSNA (ΔMAP 13 ± 3 vs. 9 ± 5 mmHg; respSNA ΔAUC, sSNA: 2.5 ± 1 vs. 1.3 ± 0.7 µV s, rSNA: 4.2 ± 0.9 vs. 3.5 ± 1.4 µV s, LPK vs. Lewis, all P > 0.05). These results demonstrate that female rats with CKD exhibit heightened respSNA coupling at baseline that is further augmented by mild hypoxia, and not by hypercapnia. This mechanism may be a contributing driver of hypertension in this animal model of CKD.

Effect of upper airway fat on tongue dilation during inspiration in awake people with obstructive sleep apnea.

STUDY OBJECTIVES: To investigate the effect of upper airway fat composition on tongue inspiratory movement and obstructive sleep apnea (OSA). METHODS: Participants without or with untreated OSA underwent a 3T magnetic resonance imaging (MRI) scan. Anatomical measurements were obtained from T2-weighted images. Mid-sagittal inspiratory tongue movements were imaged using tagged MRI during wakefulness. Tissue volumes and percentages of fat were quantified using an mDIXON scan. RESULTS: Forty predominantly overweight participants with OSA were compared to 10 predominantly normal weight controls. After adjusting for age, BMI, and gender, the percentage of fat in the tongue was not different between groups (analysis of covariance [ANCOVA], p = 0.45), but apnoeic patients had a greater tongue volume (ANCOVA, p = 0.025). After adjusting for age, BMI, and gender, higher OSA severity was associated with larger whole tongue volume (r = 0.51, p < 0.001), and greater dilatory motion of the anterior horizontal tongue compartment (r = -0.33, p = 0.023), but not with upper airway fat percentage. Higher tongue fat percentage was associated with higher BMI and older age (Spearman r = 0.43, p = 0.002, and r =0.44, p = 0.001, respectively), but not with inspiratory tongue movements. Greater inspiratory tongue movement was associated with larger tongue volume (e.g. horizontal posterior compartment, r = -0.44, p = 0.002) and smaller nasopharyngeal airway (e.g. oblique compartment, r = 0.29, p = 0.040). CONCLUSIONS: Larger tongue volume and a smaller nasopharynx are associated with increased inspiratory tongue dilation during wakefulness in people with and without OSA. This compensatory response was not influenced by higher tongue fat content. Whether this is also true in more obese patient populations requires further investigation.

Mandibular advancement splint response is associated with the pterygomandibular raphe.

STUDY OBJECTIVES: To investigate whether the presence of tendinous PMR could predict treatment outcome and how it affects lateral wall mechanical properties. Mandibular advancement increases the lateral dimensions of the nasopharyngeal airway via a direct connection from the airway to the ramus of the mandible. The anatomical structure in this region is the pterygomandibular raphe (PMR), but a tendinous component is not always present. Whether tendon presence influences treatment outcome is unknown. METHODS: In total, 105 participants with obstructive sleep apnea completed detailed anatomical magnetic resonance imaging with and without mandibular advancement. The study design was case-control. Variables were compared between participants with and without the tendon present. RESULTS: The amount of maximum mandibular advancement decreased when pterygomandibular tendon was present (4.0 ± 1.2 mm present versus 4.6 ± 1.4 mm absent, p = 0.04). PMR tendon-absent participants had a lower posttreatment apnea hypopnea index (16 ± 12 events/hour tendon present versus 9 ± 9 events/hour absent, p = 0.007) and were more likely to have complete response (63% versus 36%, p = 0.02). However, tendon-absent participants were more likely to not complete the study (χ 2 (3) = 10.578, p = 0.014). Tendon-absent participants had a greater increase in midline anteroposterior airway diameter (1.6 ± 1.7 mm versus 0.6 ± 2.3 mm, p = 0.04). CONCLUSION: When PMR tendon is absent, treatment response and amount of maximum advancement improve, possibly at the expense of reduced splint tolerability. Tendon presence may help predict a group less likely to respond to mandibular advancement splint therapy.