Molecular cell types as functional units of the efferent vagus nerve.

The vagus nerve vitally connects the brain and body to coordinate digestive, cardiorespiratory, and immune functions. Its efferent neurons, which project their axons from the brainstem to the viscera, are thought to comprise "functional units" - neuron populations dedicated to the control of specific vagal reflexes or organ functions. Previous research indicates that these functional units differ from one another anatomically, neurochemically, and physiologically but have yet to define their identity in an experimentally tractable way. However, recent work with genetic technology and single-cell genomics suggests that genetically distinct subtypes of neurons may be the functional units of the efferent vagus. Here we review how these approaches are revealing the organizational principles of the efferent vagus in unprecedented detail.

Exploring the neural mechanisms underlying achalasia: A study of functional connectivity and regional brain activity.

BACKGROUND AND AIMS: The pathophysiology of achalasia, which involves central nuclei abnormalities, remains unknown. We investigated the resting-state functional MRI (rs-fMRI) features of patients with achalasia. METHODS: We applied resting-state functional MRI (rs-fMRI) to investigate the brain features in patients with achalasia (n = 27), compared to healthy controls (n = 29). Focusing on three regions of interest (ROIs): the dorsal motor nucleus of the vagus (DMV), the nucleus ambiguus (NA), and the nucleus of the solitary tract (NTS), we analyzed variations in resting-state functional connectivity (rs-FC), fractional amplitude of low-frequency fluctuations (fALFF), and regional homogeneity (ReHo). RESULTS: Achalasia patients demonstrated stronger functional connectivity between the NA and the right precentral gyrus, left postcentral gyrus, and left insula. No significant changes were found in the DMV or NTS. The fMRI analysis showed higher rs-FC values for NA-DMV and NA-NTS connections in achalasia patients. Achalasia patients exhibited decreased fALFF values in the NA, DMV, and NTS regions, as well as increased ReHo values in the NA and DMV regions. A positive correlation was observed between fALFF values in all six ROIs and the width of the barium meal. The NTS fALFF value and NA ReHo value displayed a positive correlation with integrated relaxation pressure (IRP), while the ReHo value in the right precentral gyrus showed an inverse correlation with the height of the barium meal. CONCLUSIONS: Abnormal rs-FC and regional brain activity was found in patients with achalasia. Our study provides new insights into the pathophysiology of achalasia and highlights the potential of rs-fMRI in improving the diagnosis and treatment of this condition.

Ovariectomy Exacerbates Acute Ethanol-Induced Tachycardia: Role of Nitric Oxide and NMDA Receptors in the Rostral Ventrolateral Medulla.

Ethanol consumption influences cardiovascular functions. In humans, acute consumption of ethanol causes dose-dependent tachycardia. Our previous study showed that ethanol-induced tachycardia might involve decreased nitric oxide (NO) signaling in the brain's medulla. NMDA receptors, another important target of ethanol, are one of the upstream signals of nitric oxide. Reports showed the modulation of NMDA receptor function by estrogen or estrogen receptors. The present study aims to examine the hypothesis that depletion of estrogen by ovariectomy (OVX) might modulate ethanol-induced tachycardia by regulating NMDA receptor function and NO signaling in the cardiovascular regulatory nucleus of the brain. Ethanol (3.2 g/kg, 40% v/v, 10 mL/kg) or saline (10 mL/kg) was administered by oral gavage in sham or OVX female Sprague-Dawley (SD) rats. The blood pressure (BP) and heart rate (HR) were measured using the tail-cuff method. The levels of phosphoserine 896 of the GluN1 subunit (pGluN1-serine 896) and NMDA GluN1 subunits (GluN1) were determined by immunohistochemistry. The expressions of nitric oxide synthase (NOS) and estrogen receptors in the tissue were measured by Western blotting. Nitric oxide contents were measured as total nitrate-nitrite by colorimetric assay kit. In a 2-h observation, there was no significant change in BP between the saline and ethanol groups. However, compared with saline, ethanol caused an increase in HR (tachycardia) in sham control or OVX rats. Interestingly, ethanol produced more significant tachycardia in the OVX group than in the sham control group. Nitric oxide levels were lower in the area of the rostral ventrolateral medulla (RVLM) 60 min following ethanol administration in OVX compared with sham control, without significant changes in the expression of NOS and estrogen receptors (ERα and ERß). In addition, a decrease in the immunoreactivity of pGluN1-serine 896, without significant changes in GluN1, was found in neurons of RVLM 40 min following ethanol administration in OVX compared with sham control. Our results suggest that depletion of estradiol (E2) by OVX might exacerbate the tachycardia following ethanol administration, the underlying mechanism of which might be associated with decreased NMDA receptor function and NO level in the RVLM.

Neuroanatomical and neurophysiological evidence of pulmonary nociceptor and carotid chemoreceptor convergence in the nucleus tractus solitarius and nucleus ambiguus.

Pulmonary vagal nociceptors defend the airways. Cardiopulmonary vagal nociceptors synapse in the nucleus tractus solitarius (NTS). Evidence has demonstrated the convergence of cardiopulmonary nociceptors with afferents from carotid chemoreceptors. Whether sensory convergence occurs in motor nuclei and how sensory convergence affects reflexive efferent motor output directed toward the airways are critical knowledge gaps. Here, we show that distinct tracer injection into the pulmonary nociceptors and carotid chemoreceptors leads to co-labeled neurons in the nucleus tractus solitarius and nucleus ambiguus. Precise simultaneous stimulation delivered to pulmonary nociceptors and carotid chemoreceptors doubled efferent vagal output, enhanced phrenic pause, and subsequently augmented phrenic motor activity. These results suggest that multiple afferents are involved in protecting the airways and concurrent stimulation enhances airway defensive reflex output.NEW & NOTEWORTHY Sensory afferents have been shown to converge onto nucleus tractus solitarius primary neurons. Here, we show sensory convergence of two distinct sets of sensory afferents in motor nuclei of the nucleus ambiguus, which results in augmentation of airway defense motor output.

Heightened respiratory-parasympathetic coupling to airways in the spontaneously hypertensive rat.

KEY POINTS: Carotid body (CB) chemoreceptors are hyperactive in hypertension, and their acute activation produces bronchoconstriction. We show that the respiratory-modulated bronchiolar tone, pulmonary parasympathetic efferent activity, and the firing frequency and synaptic excitation of bronchoconstrictor motoneurones in the nucleus ambiguus were all enhanced in spontaneous hypertensive (SH) rats. In SH rats, CB denervation reduced the respiratory-related parasympathetic-mediated bronchoconstrictor tone to levels seen in normotensive rats. Chemoreflex evoked bronchoconstrictor tone was heightened in SH versus normotensive rats. The intrinsic electrophysiological properties and morphology of bronchoconstrictor motoneurones were similar across rat strains. The heightened respiratory modulation of parasympathetic-mediated bronchoconstrictor tone to the airways in SH rats is caused by afferent drive from the CBs. ABSTRACT: Much research has described heightened sympathetic activity in hypertension and diminished parasympathetic tone, especially to the heart. The carotid body (CB) chemoreceptors exhibit hyperreflexia and are hyperactive, providing excitatory drive to sympathetic networks in hypertension. Given that acute CB activation produces reflex evoked bronchoconstriction via activation of parasympathetic vagal efferents, we hypothesised that the parasympathetic bronchoconstrictor activity is enhanced in spontaneously hypertensive (SH) rats and that this is dependent on CB inputs. In situ preparations of Wistar and SH rats were used in which bronchiolar tone, the pulmonary branch of the vagus (pVN) and phrenic nerves were recorded simultaneously; whole cell patch clamp recordings of bronchoconstrictor vagal motoneurones were also made from the nucleus ambiguus. Bronchiolar tone, pVN and bronchoconstrictor motoneurones were respiratory modulated and this modulation was enhanced in SH rats. These differences were all eliminated after CB denervation. Stimulation of the CBs increased the phrenic frequency that caused a summation of the respiratory-related increases in pVN, resulting in the development of bronchoconstrictor tone. This tone was exaggerated in SH rats. The enhanced respiratory-parasympathetic coupling to airways in SH rats was not due to differences in the intrinsic electrophysiological properties of bronchoconstrictor motoneurones but reflected heightened pre-inspiratory- and inspiratory-related synaptic drive. In summary, in SH rats the phasic respiratory modulation of parasympathetic tone to the airways is elevated and the greater development of this bronchoconstrictor tone is caused by the heightened afferent drive originating from the CBs. Thus, targeting the CBs may prove effective for increasing lower airway patency.

Adrenergic agonist induces rhythmic firing in quiescent cardiac preganglionic neurons in nucleus ambiguous via activation of intrinsic membrane excitability.

Cholinergic vagal nerves projecting from neurons in the brain stem nucleus ambiguus (NAm) play a predominant role in cardiac parasympathetic pacemaking control. Central adrenergic signaling modulates the tone of this vagal output; however, the exact excitability mechanisms are not fully understood. We investigated responses of NAm neurons to adrenergic agonists using in vitro mouse brain stem slices. Preganglionic NAm neurons were identified by ChAT-tdTomato fluorescence in young adult transgenic mice, and their cardiac projection was confirmed by retrograde dye tracing. Juxtacellular recordings detected sparse or absent spontaneous action potentials (AP) in NAm neurons. However, bath application of epinephrine or norepinephrine strongly and reversibly activated most NAm neurons regardless of their basal firing rate. Epinephrine was more potent than norepinephrine, and this activation largely depends on α1-adrenoceptors. Interestingly, adrenergic activation of NAm neurons does not require an ionotropic synaptic mechanism, because postsynaptic excitatory or inhibitory receptor blockade did not occlude the excitatory effect, and bath-applied adrenergic agonists did not alter excitatory or inhibitory synaptic transmission. Instead, adrenergic agonists significantly elevated intrinsic membrane excitability to facilitate generation of recurrent action potentials. T-type calcium current and hyperpolarization-activated current are involved in this excitation pattern, although not required for spontaneous AP induction by epinephrine. In contrast, pharmacological blockade of persistent sodium current significantly inhibited the adrenergic effects. Our results demonstrate that central adrenergic signaling enhances the intrinsic excitability of NAm neurons and that persistent sodium current is required for this effect. This central balancing mechanism may counteract excessive peripheral cardiac excitation during increased sympathetic tone. NEW & NOTEWORTHY Cardiac preganglionic cholinergic neurons in the nucleus ambiguus (NAm) are responsible for slowing cardiac pacemaking. This study identified that adrenergic agonists can induce rhythmic action potentials in otherwise quiescent cholinergic NAm preganglionic neurons in brain stem slice preparation. The modulatory influence of adrenaline on central parasympathetic outflow may contribute to both physiological and deleterious cardiovascular regulation.

Characteristics of single large-conductance Ca2+-activated K+ channels and their regulation of action potentials and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus.

Large-conductance Ca2(+)-activated K+ channels (BK) regulate action potential (AP) properties and excitability in many central neurons. However, the properties and functional roles of BK channels in parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA) have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal 7-9 days. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. Using excised patch single-channel recordings, we identified voltage-gated and Ca(2+)-dependent BK channels in PCMNs. The majority of BK channels exhibited persistent channel opening during voltage holding. These BK channels had a conductance of 237 pS and a 50% opening probability at +27.9 mV, the channel open time constant was 3.37 ms at +20 mV, and dwell time increased exponentially as the membrane potential depolarized. At the +20-mV holding potential, the [Ca2+]50 was 15.2 µM with a P0.5 of 0.4. Occasionally, some BK channels showed a transient channel opening and fast inactivation. Using whole cell voltage clamp, we found that BK channel mediated outward currents and afterhyperpolarization currents (IAHP). Using whole cell current clamp, we found that application of BK channel blocker iberiotoxin (IBTX) increased spike half-width and suppressed fast afterhyperpolarization (fAHP) amplitude following single APs. In addition, IBTX application increased spike half-width and reduced the spike frequency-dependent AP broadening in trains and spike frequency adaption (SFA). Furthermore, BK channel blockade decreased spike frequency. Collectively, these results demonstrate that PCMNs have BK channels that significantly regulate AP repolarization, fAHP, SFA, and spike frequency. We conclude that activation of BK channels underlies one of the mechanisms for facilitation of PCMN excitability.

Urotensin II promotes vagal-mediated bradycardia by activating cardiac-projecting parasympathetic neurons of nucleus ambiguus.

Urotensin II (U-II) is a cyclic undecapeptide that regulates cardiovascular function at central and peripheral sites. The functional role of U-II nucleus ambiguus, a key site controlling cardiac tone, has not been established, despite the identification of U-II and its receptor at this level. We report here that U-II produces an increase in cytosolic Ca(2+) concentration in retrogradely labeled cardiac vagal neurons of nucleus ambiguus via two pathways: (i) Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptor; and (ii) Ca(2+) influx through P/Q-type Ca(2+) channels. In addition, U-II depolarizes cultured cardiac parasympathetic neurons. Microinjection of increasing concentrations of U-II into nucleus ambiguus elicits dose-dependent bradycardia in conscious rats, indicating the in vivo activation of the cholinergic pathway controlling the heart rate. Both the in vitro and in vivo effects were abolished by the urotensin receptor antagonist, urantide. Our findings suggest that, in addition, to the previously reported increase in sympathetic outflow, U-II activates cardiac vagal neurons of nucleus ambiguus, which may contribute to cardioprotection.

Evidence that BDNF regulates heart rate by a mechanism involving increased brainstem parasympathetic neuron excitability.

Autonomic control of heart rate is mediated by cardioinhibitory parasympathetic cholinergic neurons located in the brainstem and stimulatory sympathetic noradrenergic neurons. During embryonic development the survival and cholinergic phenotype of brainstem autonomic neurons is promoted by brain-derived neurotrophic factor (BDNF). We now provide evidence that BDNF regulates heart rate by a mechanism involving increased brainstem cardioinhibitory parasympathetic activity. Mice with a BDNF haploinsufficiency exhibit elevated resting heart rate, and infusion of BDNF intracerebroventricularly reduces heart rate in both wild-type and BDNF+/- mice. The atropine-induced elevation of heart rate is diminished in BDNF+/- mice and is restored by BDNF infusion, whereas the atenolol-induced decrease in heart rate is unaffected by BDNF levels, suggesting that BDNF signaling enhances parasympathetic tone which is diminished with BDNF haploinsufficiency. Whole-cell recordings from pre-motor cholinergic cardioinhibitory vagal neurons in the nucleus ambiguus indicate that BDNF haploinsufficiency reduces cardioinhibitory vagal neuron activity by increased inhibitory GABAergic and diminished excitatory glutamatergic neurotransmission to these neurons. Our findings reveal a previously unknown role for BDNF in the control of heart rate by a mechanism involving increased activation of brainstem cholinergic parasympathetic neurons.

HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats.

The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1-86) produced a concentration-dependent increase in cytosolic Ca(2+) in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca(2+) elevation: 1) lysosomal Ca(2+) mobilization, 2) Ca(2+) release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca(2+) influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca(2+) entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.

Microinjection of l-glutamate into the nucleus ambiguus partially inhibits gastric motility through the NMDA receptor - nitric oxide pathway.

We have previously reported that both l-glutamate (l-Glu) and nitric oxide (NO) modulate gastric motility in the nucleus ambiguus (NA). The aim of this study is to explore the potential correlation between the l-Glu and NO. A latex balloon connected to a pressure transducer was inserted into the pylorus through the fundus of anesthetized male Wistar rats to continuously record changes in gastric smooth muscle contractile curves. Pretreatment with the NO-synthase inhibitor N-nitro-l-arginine methylester (l-NAME) did not completely abolish the inhibitory effect of l-Glu on gastric motility, but intravenous injection of the ganglionic blocker hexamethonium bromide (Hb) did. By using a specific N-methyl-d-aspartic acid (NMDA) receptor antagonist, we blocked the inhibitory effect of the NO-donor sodium nitroprusside (SNP) on gastric motility. These results suggest that microinjections of l-Glu into the NA inhibits gastric motility by activating the cholinergic preganglionic neurons, partially through the NMDA receptor - NO pathway.

Investigating the L-Glu-NMDA receptor-H2S-NMDA receptor pathway that regulates gastric function in rats' nucleus ambiguus.

Background: In previous investigations, we explored the regulation of gastric function by hydrogen sulfide (H2S) and L-glutamate (L-Glu) injections in the nucleus ambiguus (NA). We also determined that both H2S and L-Glu have roles to play in the physiological activities of the body, and that NA is an important nucleus for receiving visceral sensations. The purpose of this study was to explore the potential pathway link between L-Glu and H2S, resulting in the regulation of gastric function. Methods: Physiological saline (PS), L-glutamate (L-Glu, 2 nmol), NaHS (2 nmol), D-2-amino-5-phopho-novalerate (D-AP5, 2 nmol) + L-Glu (2 nmol), aminooxyacetic acid (AOAA, 2 nmol) + L-Glu (2 nmol), D-AP5 (2 nmol) + NaHS (2 nmol) were injected into the NA. A balloon was inserted into the stomach to observe gastric pressure and for recording the changes of gastric smooth muscle contraction curve. The gastric fluid was collected by esophageal perfusion and for recording the change of gastric pH value. Results: Injecting L-Glu in NA was found to significantly inhibit gastric motility and promote gastric acid secretion in rats (p < 0.01). On the other hand, injecting the PS, pre-injection N-methyl-D-aspartate (NMDA) receptor blocker D-AP5, cystathionine beta-synthase (CBS) inhibitor AOAA and re-injection L-Glu did not result in significant changes (p > 0.05). The same injection NaHS significantly inhibit gastric motility and promote gastric acid secretion in rats (p < 0.01), but is eliminated by injection D-AP5 (p > 0.05). Conclusion: The results indicate that both exogenous L-Glu and H2S injected in NA regulate gastric motility and gastric acid secretion through NMDA receptors. This suggests that NA has an L-Glu-NMDA receptor-CBS-H2S pathway that regulates gastric function.

Visualizing oxidative stress-induced depression of cardiac vagal baroreflex by MRI/DTI in a mouse neurogenic hypertension model.

A clinical hallmark of hypertension is impairment of the cardiac vagal baroreflex, which maintains stable blood pressure and heart rate under physiological conditions. There is also evidence that oxidative stress in the brain is associated with neurogenic hypertension. We tested the hypothesis that an augmented superoxide level in the nucleus tractus solitarii (NTS), the terminal site of baroreceptor afferents, contributes to the depression of cardiac vagal baroreflex by disrupting the connectivity between the NTS and the nucleus ambiguus (NA), the origin of the vagus nerve, during neurogenic hypertension. An experimental model of neurogenic hypertension that employed intracerebroventricular infusion of angiotensin II in male adult C57BL/6 mice was used. Based on tractographic evaluations using magnetic resonance imaging/diffusion tensor imaging of the medulla oblongata in the brain stem, we found that the connectivity between the NTS and NA was disrupted in neurogenic hypertension, concurrent with impairment of the cardiac vagal baroreflex as detected by radiotelemetry. We further found that the disrupted NTS-NA connectivity was reversible, and was related to oxidative stress induced by augmented levels of NADPH oxidase-generated superoxide in the NTS. We conclude that depression of the cardiac vagal baroreflex induced by oxidative stress in the NTS in the context of neurogenic hypertension may be manifested in the form of dynamic alterations in the connectivity between the NTS and NA.

Expression of Glial Cell-Derived Neurotrophic Factor Receptors Within Nucleus Ambiguus During Rat Development.

OBJECTIVE: The nucleus ambiguus (NAmb) is a column of neurons in the medulla oblongata, involved in bulbar functions. Expression of Glial Cell-Derived Neurotrophic Factor (GDNF) and its receptors (GDNFR) is observed within the cell bodies during reinnervation following recurrent laryngeal nerve (RLN) injury. Little is known regarding GDNFR expression in the formation of the NAmb and the laryngeal innervation during embryogenesis. Understanding the timing and pattern of GDNFR expression in embryogenesis versus after RLN injury may provide insights into therapeutic targets for regeneration after RLN injury. STUDY DESIGN: Laboratory experiment. METHODS: Rat brainstems at E14.5/E16.5/E18.5/E20.5/adult were stained for GDNFR: GFRα-1/GFRα-2/GFRα-3/Ret. Islet1 and choline acetyltransferase were used as cell body markers. Sections were observed using fluorescent microscopy and quantified through manual cell counting. RESULTS: Expression of GFRα-1, GFRα-3, and Ret was identified within the NAmb, hypoglossal, and facial nuclei of the adult medulla. During development, GFRα-1 immunoreactivity was seen at E20.5. GFRα-2 expression was not observed at any timepoint. GFRα-3 expression began at E16.5. Ret expression within nerve fibers in the NAmb were observed beginning at E14.5, but never in the cell bodies. CONCLUSION: Embryonic GDNFR expression in the NAmb differs from that of the adult after RLN injury. The developing brainstem experienced upregulation at discrete timepoints with signaling sustained through adulthood. In contrast, adult RLN-transected rats experienced patterns of up and down regulation. GFRα-1 may contribute to muscle targeting and neuromuscular junction maturation, GFRα-3 may contribute to both, as well as axon guidance. It is likely that GDNF is functioning via a Ret-independent pathway. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2240-2247, 2023.

Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation.

OBJECTIVES: Recurrent laryngeal nerve (RLN) injury results in synkinetic reinnervation and vocal fold paralysis. Investigation of cues expressed in the developing brainstem that influence correct selective targeting of intrinsic laryngeal muscles may elucidate post-injury abnormalities contributing to non-functional reinnervation. Primary targets of interest were Hoxb1 and Hoxb2, members of the Hox family that create overlapping gradients in the developing brain, and their target Phox2b, a transcription factor necessary for cranial nerve branchio- and visceromotoneuron survival. METHODS: Rat embryos at developmental days E14, E16, E18, and E20 (4 animals/age) were sectioned for RNA in situ hybridization to detect Hoxb1, Hoxb2, and Phox2b mRNA within the brainstem. Slides were costained with Islet1 antibody for identification of the nucleus ambiguus. Results were confirmed using immunohistochemistry. Sections were imaged on a confocal microscope. RNA and protein expressions were quantified using QuPath. Statistical analyses were performed using R. RESULTS: Hoxb1, Hoxb2, and Phox2b expressions varied according to embryologic age. Hoxb1 and Hoxb2 expression peaked at E16, with significant decreases at E18 and E20 (one-way ANOVA p = 0.001 for both). Phox2b expression was highest at E14 and trended downward with increased embryologic age (one-way ANOVA p = 0.005). CONCLUSION: Peak expression of Hoxb1 and Hoxb2 is observed at time points when the RLN arrives at the larynx and begins to branch toward individual muscles, positioning these gene products to be involved in cueing laryngeal motoneuron identity and target identification. Higher expression of Phox2b earlier in development suggests a role in laryngeal motoneuron formation. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3462-3471, 2023.

Fundamental challenges and likely refutations of the five basic premises of the polyvagal theory.

The polyvagal collection of hypotheses is based upon five essential premises, as stated by its author (Porges, 2011). Polyvagal conjectures rest on a primary assumption that brainstem ventral and dorsal vagal regions in mammals each have their own unique mediating effects upon control of heart rate. The polyvagal hypotheses link these putative dorsal- vs. ventral-vagal differences to socioemotional behavior (e.g. defensive immobilization, and social affiliative behaviors, respectively), as well as to trends in the evolution of the vagus nerve (e.g. Porges, 2011 & 2021a). Additionally, it is essential to note that only one measurable phenomenon-as index of vagal processes-serves as the linchpin for virtually every premise. That phenomenon is respiratory sinus arrhythmia (RSA), heart-rate changes coordinated to phase of respiration (i.e. inspiration vs. expiration), often employed as an index of vagally, or parasympathetically, mediated control of heart rate. The polyvagal hypotheses assume that RSA is a mammalian phenomenon, since Porges (2011) states "RSA has not been observed in reptiles." I will here briefly document how each of these basic premises have been shown to be either untenable or highly implausible based on the available scientific literature. I will also argue that the polyvagal reliance upon RSA as equivalent to general vagal tone or even cardiac vagal tone is conceptually a category mistake (Ryle, 1949), confusing an approximate index (i.e. RSA) of a phenomenon (some general vagal process) with the phenomenon, itself.

Cardiovascular baroreflex circuit moonlights in sleep control.

Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.

Functional anatomy of the vagus system: How does the polyvagal theory comply?

Due to its pivotal role in autonomic networks and interoception, the vagus attracts continued interest from both basic scientists and therapists of various clinical disciplines. In particular, the widespread use of heart rate variability as an index of autonomic cardiac control and a proposed central role of the vagus in biopsychological concepts, e.g., the polyvagal theory, provide a good opportunity to recall basic features of vagal anatomy. In addition to the "classical" vagal brainstem nuclei, i.e., dorsal motor nucleus, nucleus ambiguus and nucleus tractus solitarii, the spinal trigeminal and paratrigeminal nuclei come into play as targets of vagal afferents. On the other hand, the nucleus of the solitary tract receives and integrates not only visceral but also somatic afferents.

Genetic encoding of an esophageal motor circuit.

Motor control of the striated esophagus originates in the nucleus ambiguus (nAmb), a vagal motor nucleus that also contains upper airway motor neurons and parasympathetic preganglionic neurons for the heart and lungs. We disambiguate nAmb neurons based on their genome-wide expression profiles, efferent circuitry, and ability to control esophageal muscles. Our single-cell RNA sequencing analysis predicts three molecularly distinct nAmb neuron subtypes and annotates them by subtype-specific marker genes: Crhr2, Vipr2, and Adcyap1. Mapping the axon projections of the nAmb neuron subtypes reveals that Crhr2nAmb neurons innervate the esophagus, raising the possibility that they control esophageal muscle function. Accordingly, focal optogenetic stimulation of cholinergic Crhr2+ fibers in the esophagus results in contractions. Activating Crhr2nAmb neurons has no effect on heart rate, a key parasympathetic function of the nAmb, whereas activating all of the nAmb neurons robustly suppresses heart rate. Together, these results reveal a genetically defined circuit for motor control of the esophagus.

The multibranched nerve: vagal function beyond heart rate variability.

This paper reviews the many functions of the vagus nerve, to understand how they interact in daily life and what might be accomplished by therapeutical electrical stimulation. A short historical introduction on the discovery and name-giving of the cranial nerves numbers 9-12 is followed by an overview of the functions that are under lower brain stem control: heart (rate, contractility), intestine (swallowing, peristalsis and glands secretions, feeling of satiety), lungs (bronchoconstriction, lung-irritant and stretch receptor signaling), blood pressure (by vascular wall stress sensing) and blood gases by specialized receptors. Key in the review is the physiology behind beat-by-beat heart rate variations, how everyday life is reflected in its variability, from exciting moments to quiet sleep, with the 'common faint' or vasovagal collapse as extreme example. Next, the recently proposed role of the vagus nerve in limiting inflammation is discussed. This has led to adoption of an earlier developed technique for epilepsy treatment, i.e., electrical stimulation of one vagus nerve bundle in the neck, but now for immune diseases like rheumatoid arthritis and the scope is even widening to depression and cluster headache. However, the problem in application of whole vagus nerve stimulation is the lack of specificity: there is no way to titrate the stimulation to an observable effect variable. All nerves in the bundle, incoming and outgoing, can be 'hit', leading to side-effects which limit the intended application.