Nucleus Ambiguus Research Articles

Immediate and sustained increases in the activity of vagal preganglionic neurons during exercise and after exercise training.

The brain controls the heart by dynamic recruitment and withdrawal of cardiac parasympathetic (vagal) and sympathetic activity. Autonomic control is essential for the development of cardiovascular responses during exercise, however, the patterns of changes in the activity of the two autonomic limbs, and their functional interactions in orchestrating physiological responses during exercise, are not fully understood. The aim of this study was to characterize changes in vagal parasympathetic drive in response to exercise and exercise training by directly recording the electrical activity of vagal preganglionic neurons in experimental animals (rats). Single unit recordings were made using carbon-fibre microelectrodes from the populations of vagal preganglionic neurons of the nucleus ambiguus (NA) and the dorsal vagal motor nucleus of the brainstem. It was found that (i) vagal preganglionic neurons of the NA and the dorsal vagal motor nucleus are strongly activated during bouts of acute exercise, and (ii) exercise training markedly increases the resting activity of both populations of vagal preganglionic neurons and augments the excitatory responses of NA neurons during exercise. These data show that central vagal drive increases during exercise and provide the first direct neurophysiological evidence that exercise training increases vagal tone. The data argue against the notion of exercise-induced central vagal withdrawal during exercise. We propose that robust increases in the activity of vagal preganglionic neurons during bouts of exercise underlie activity-dependent plasticity, leading to higher resting vagal tone that confers multiple health benefits associated with regular exercise.

Sodium appetite and thirst do not require angiotensinogen production in astrocytes or hepatocytes.

In addition to its renal and cardiovascular functions, angiotensin signaling is thought to be responsible for the increases in salt and water intake caused by hypovolemia. However, it remains unclear whether these behaviors require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, then use conditional genetic deletion of angiotensinogen (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor of all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (enzymes required to produce angiotensin II) in the choroid plexus, and Ren1 in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is expressed in widespread hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signaling is not required for sodium appetite or thirst and highlight the need to identify alternative signaling mechanisms. KEY POINTS: Angiotensin signaling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiologic mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolemic signals necessary for activating each behavior. Abstract figure legend Angiotensinogen is the precursor for angiotensin II, which is considered important for thirst and sodium appetite. In the brain, astrocytes express the angiotensinogen gene (Agt), but using GFAP-Cre to delete this gene from astrocytes did not reduce thirst or sodium appetite. In the liver, hepatocytes express this gene and produce circulating angiotensinogen. Using Alb-Cre to delete Agt from hepatocytes also did not reduce (and in fact enhanced) salt and water consumption. This article is protected by copyright. All rights reserved.

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

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. 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. 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). 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. NA Laryngoscope, 133:3462-3471, 2023.

Galantamine ameliorates hyperoxia-induced brain injury in neonatal mice.

Prolonged oxygen therapy in preterm infants often leads to cognitive impairment. Hyperoxia leads to excess free radical production with subsequent neuroinflammation, astrogliosis, microgliosis and apoptosis. We hypothesized that Galantamine, an acetyl choline esterase inhibitor and an FDA approved treatment of Alzheimer's disease, will reduce hyperoxic brain injury in neonatal mice and will improve learning and memory. Mouse pups at postnatal day 1 (P1) were placed in a hyperoxia chamber (FiO2 95%) for 7 days. Pups were injected IP daily with Galantamine (5 mg/kg/dose) or saline for 7 days. Hyperoxia caused significant neurodegeneration in cholinergic nuclei of the basal forebrain cholinergic system (BFCS), laterodorsal tegmental (LDT) nucleus and nucleus ambiguus (NA). Galantamine ameliorated this neuronal loss. Treated hyperoxic group showed a significant increase of choline acetyl transferase (ChAT) expression and a decrease of acetyl choline esterase activity, thus increasing acetyl choline levels in hyperoxia environment. Hyperoxia increased pro-inflammatory cytokines namely IL -1β, IL-6 and TNF α, HMGB1, NF-κB activation. Galantamine showed its potent anti- inflammatory effect, by blunting cytokines surges among treated group. Treatment with Galantamine increased myelination while reducing apoptosis, microgliosis, astrogliosis and ROS production. Long term neurobehavioral outcomes at P60 showed improved locomotor activity, coordination, learning and memory, along with increased hippocampal volumes on MRI with Galantamine treated versus non treated hyperoxia group. Together our findings suggest a potential therapeutic role for Galantamine in attenuating hyperoxia-induced brain injury.

Whole-brain monosynaptic inputs and outputs of leptin receptor b neurons of the nucleus tractus solitarii in mice

The nucleus tractus solitarii (NTS) is the primary central station that integrates visceral afferent information and regulates respiratory, gastrointestinal, cardiovascular, and other physiological functions. Leptin receptor b (LepRb)-expressing neurons of the NTS (NTSLepRb neurons) are implicated in central respiration regulation, respiratory facilitation, and respiratory drive enhancement. Furthermore, LepRb dysfunction is involved in obesity, insulin resistance, and sleep-disordered breathing. However, the monosynaptic inputs and outputs of NTSLepRb neurons in whole-brain mapping remain to be elucidated. Therefore, the exploration of its whole-brain connection system may provide strong support for comprehensively understanding the physiological and pathological functions of NTSLepRb neurons. In the present study, we used a cell type-specific, modified rabies virus and adeno-associated virus with the Cre-loxp system to map monosynaptic inputs and outputs of NTSLepRb neurons in LepRb-Cre mice. The results showed that NTSLepRb neurons received inputs from 48 nuclei in the whole brain from five brain regions, including especially the medulla. We found that NTSLepRb neurons received inputs from nuclei associated with respiration, such as the pre-Bötzinger complex, ambiguus nucleus, and parabrachial nucleus. Interestingly, some brain areas related to cardiovascular regulation—i.e., the ventrolateral periaqueductal gray and locus coeruleus—also sent a small number of inputs to NTSLepRb neurons. In addition, anterograde tracing results demonstrated that NTSLepRb neurons sent efferent projections to 15 nuclei, including the dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus, which are involved in regulation of energy metabolism and feeding behaviors. Quantitative statistical analysis revealed that the inputs of the whole brain to NTSLepRb neurons were significantly greater than the outputs. Our study comprehensively revealed neuronal connections of NTSLepRb neurons in the whole brain and provided a neuroanatomical basis for further research on physiological and pathological functions of NTSLepRb neurons.

Fentanyl-Induced Respiratory Depression and Locomotor Hyperactivity Are Mediated by μ-Opioid Receptors Expressed in Somatostatin-Negative Neurons

AbstractOpioid drugs are widely used as analgesics but cause respiratory depression, a potentially lethal side effect with overdose, by acting on μ-opioid receptors (MORs) expressed in brainstem regions involved in the control of breathing. Although many brainstem regions have been shown to regulate opioid-induced respiratory depression, the types of neurons involved have not been identified. Somatostatin is a major neuropeptide found in brainstem circuits regulating breathing, but it is unknown whether somatostatin-expressing circuits regulate respiratory depression by opioids. We examined the coexpression ofSst(gene encoding somatostatin) andOprm1(gene encoding MORs) mRNAs in brainstem regions involved in respiratory depression. Interestingly,Oprm1mRNA expression was found in the majority (>50%) ofSst-expressing cells in the preBötzinger Complex, the nucleus tractus solitarius, the nucleus ambiguus, and the Kölliker–Fuse nucleus. We then compared respiratory responses to fentanyl between wild-type andOprm1full knock-out mice and found that the lack of MORs prevented respiratory rate depression from occurring. Next, using transgenic knock-out mice lacking functional MORs specifically inSst-expressing cells, we compared respiratory responses to fentanyl between control and the conditional knock-out mice. We found that respiratory rate depression by fentanyl was preserved when MORs were deleted only inSst-expressing cells. Our results show that despite coexpression ofSstandOprm1in respiratory circuits and the importance of somatostatin-expressing cells in the regulation of breathing, these cells do not mediate opioid-induced respiratory rate depression. Instead, MORs found in respiratory cell populations other thanSst-expressing cells likely contribute to the respiratory effects of fentanyl.

Two conserved vocal central pattern generators broadly tuned for fast and slow rates generate species-specific vocalizations in Xenopus clawed frogs.

Across phyla, males often produce species-specific vocalizations to attract females. Although understanding the neural mechanisms underlying behavior has been challenging in vertebrates, we previously identified two anatomically distinct central pattern generators (CPGs) that drive the fast and slow clicks of male Xenopus laevis, using an ex vivo preparation that produces fictive vocalizations. Here, we extended this approach to four additional species, X. amieti, X. cliivi, X. petersii, and X. tropicalis, by developing ex vivo brain preparation from which fictive vocalizations are elicited in response to a chemical or electrical stimulus. We found that even though the courtship calls are species-specific, the CPGs used to generate clicks are conserved across species. The fast CPGs, which critically rely on reciprocal connections between the parabrachial nucleus and the nucleus ambiguus, are conserved among fast-click species, and slow CPGs are shared among slow-click species. In addition, our results suggest that testosterone plays a role in organizing fast CPGs in fast-click species, but not in slow-click species. Moreover, fast CPGs are not inherited by all species but monopolized by fast-click species. The results suggest that species-specific calls of the genus Xenopus have evolved by utilizing conserved slow and/or fast CPGs inherited by each species.

Topographic Consideration on the Occurrence of Ipsilesional Facial Paresis in Lateral Medullary Infarction

Introduction: The purpose of this study was to identify course of the corticobulbar tract and factors associated with the occurrence of facial paresis (FP) in lateral medullary infarction (LMI). Methods: Patients diagnosed with LMI who were admitted to tertiary hospital were retrospectively investigated and divided into two groups based on the presence of FP. FP was defined as grade 2 or more by the House-Brackmann scale. Differences between the two groups were analyzed with respect to anatomical location of the lesions, demographic data (age, sex), risk factors (diabetes, hypertension, smoking, prior stroke, atrial fibrillation, and other cardiac risk factors for stroke), large vessel involvement on magnetic resonance angiography, other symptoms and signs (sensory symptoms, gait ataxia, limb ataxia, dizziness, Horner syndrome, hoarseness, dysphagia, dysarthria, nystagmus, nausea/vomiting, headache, neck pain, diplopia, and hiccup). Results: Among 44 LMI patients, 15 patients (34%) had FP, and all of them had ipsilesional central-type FP. The FP group tended to involve upper (p < 0.0001) and relative ventral (p = 0.019) part of the lateral medulla. Horizontally large lesion was also related to the presence of FP (p = 0.044). Dysphagia (p = 0.001), dysarthria (p = 0.003), and hiccups (p = 0.034) were more likely to be accompanied by FP. Otherwise, there were no significant differences. Conclusion: The results of present study indicate that the corticobulbar fibers innervating the lower face decussate at the upper level of the medulla and ascend through the dorsolateral medulla, where the concentration of the fibers is densest near the nucleus ambiguus.

Central Autonomic Nervous System Balanced by Electroacupuncture in Hypertension

Overexcitation of sympathetic activity strongly related to increased blood pressure (BP) is decreased by electroacupuncture (EA). Reflex elevated BP response and elevated neuronal activity in the rostral ventrolateral medulla (rVLM), a central region for sympathetic outflow are reduced by stimulation of somatosensory nerves during a 30 min EA. However, it is unclear if the medullary nucleus ambiguus (NA) related to parasympathetic activity also is influenced by EA during elevated BP. In hypertensive subjects, EA therapy over deep peroneal and tibial nerves (ST36-37+SP6-7) decreases high BP. We hypothesize that a course of EA treatments decreases high BP by reducing rVLM and simultaneously increasing NA neuronal activity in hypertensive rats. Dahl salt-sensitive rats were fed either a standard rat diet or 4% salt rat diet developing hypertension. Normotensive control rats were acclimated to handling and restraining while Sham-EA (absence of electrical input to acupuncture needles) and EA treated hypertensive rats were acclimatized also to needling. Sham-EA or EA was given twice a week for 30 minutes in awake hypertensive rats for 5 weeks. BPs were measured with tail-cuff CODA system. At end of 10 weeks, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) neuronal (NeuN) expressions were evaluated by immunohistochemistry as well as neuronal activity were determined by extracellular recordings in the rVLM and NA in Sham-EA, EA treated rats, and control rats. BP of control rats was at 130.3±1.7 mmHg (n=6) at end of experiment. BP significantly decreased by 25.9% in EA treated (138.04±1.8 mmHg, n=6) compared to Sham-EA (186.3±2.3 mmHg, n=7) hypertensive rats. Cardiovascular pre-sympathetic neuronal activity in rVLM significantly was decreased by 43% (1.33±0.10 to 0.75±0.13 sp/s, n=10) while the pre-parasympathetic activity in NA was increased by 96% (0.70±0.08 to 1.37±0.13 sp/s, n=12) in EA compared to Sham-EA treated hypertensive rats. Control rats rVLM activity was 0.23± 0.07 sp/s (n=4) and NA activity was 2.71±0.56 sp/s (n=5). Also, numbers of rVLM neurons expressing TH and NeuN were decreased (from 96 to 73) and NA neurons expressing ChAT and NeuN were increased (from 35 to 107) in Sham-EA compared to EA treated hypertensive rats. In normal control rats, rVLM showed 69 TH neurons and NA demonstrated 134 ChAT neurons. In conclusion, the BP lowering EA effect likely involved central autonomic rebalance by activating both sympathoinhibition and parasympathoexcitation. National Institute of Health National Center for Complementary &amp; Integrative Health Grant AT011306 This is the full abstract presented at the American Physiology Summit 2023 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.

Divergent electrophysiology between two cardiac vagal motor populations despite similar chronotropic effects

Cardiac vagal motor output is generated by brainstem cardiac vagal motorneurons (CVNs) that send axonal projections, through the vagus nerve, to cardiac ganglia. CVNs have a well-established cardioinhibitory influence through reductions in heart rate (HR) and studies using vagotomy confirm cardiac vagal drive originates in CVNs. CVNs can be found in two brainstem regions: nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV). Although the cardioinhibitory role of CVNs in NA (CVN NA ) is widely accepted, effects of CVNs in DMV (CVN DMV ) on HR regulation is controversial. The present study aims to provide evidence of CVN DMV existence and identify the unique role DMV plays in HR regulation. We hypothesized that CVN DMV cause robust bradycardias and associate with unique electrophysiological properties making them hyperexcitable compared to CVN NA . Experiments were performed on adult (9-11 week old) male mice. Using a transgenic mouse line where light activated channelrhodopsin (ChR) is expressed in choline acetyltransferase neurons, activation of ChR in DMV using fiber optic probes resulted in a 359±43 bpm bradycardia (n=8) in awake behaving animals. DMV-mediated ChR-dependent bradycardia remained after i.p. administration of atenolol (256±31 bpm). However, i.p. scopolamine-methyl nitrate abolished the ability of ChR activation of DMV to impact HR (12±2 bpm, p=0.0002). ChR stimulation of NA resulted in a similar bradycardia (447±6 bpm; p&lt;0.0001) that was also blocked by scopolamine-methyl nitrate (9±7 bpm, p=0.005). Retrograde tracing from cardiac tissue using rhodamine and cholera-toxin B (CTB) confirmed CVNs in DMV (50±15 labeled neurons, n=6; CTB: 12±4 labeled neurons, n=8). However, the number of CVN DMV was significantly lower than CVN NA (rhodamine: 116±24 labeled neurons, n=6; CTB: 83.33±9.548 labeled neurons, n=6, p&lt;0.0001 for both). Unilateral vagotomy eliminated tracer presence in DMV ipsilateral (2±1 neurons) compared to contralateral (32±8 neurons; n=4; p=0.048). Using whole cell patch clamp, CVN DMV demonstrated spontaneous action potential firing in vitro (0.7±0.3 Hz, n=14 neurons/10 mice) whereas CVN NA were devoid of spontaneous firing (0.0±0.0 Hz, n=10 neurons/9 mice). CVN DMV (-58.6±2.7 Hz, n=14 neurons/10 mice) and CVN NA (-62.2±1.8 mV, n=10 neurons/9 mice, p=0.3) demonstrated similar resting membrane potential. However, CVN DMV (492.0±22.0 MΩ, n=14 neurons/10 mice) had higher input resistance compared to CVN NA (158.2±10.6 MΩ, n=10 neurons/9 mice, p&lt;0.0001). Taken together, these data support the hypothesis that DMV contains a population of CVNs capable of producing cardioinhibitory actions with unique hyperexcitability compared to CVN NA . Future studies will examine both intracellular mechanism(s) of these differences, but also unique microcircuit pathways which utilize CVN DMV , and not CVN NA . NIH T32 HL007446 for MS; NIH R01HL157366 to CRB This is the full abstract presented at the American Physiology Summit 2023 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.

Genetic and Functional Identification of Cardiovagal Nucleus Ambiguus Neurons

Heart rate is an important physiological index of overall health and is primarily controlled by the parasympathetic nervous system, which suppresses heart rate at rest and during cardiac reflexes. The nucleus ambiguus (nAmb), a region found within the brainstem, is a primary source of parasympathetic input to the heart. It also innervates striated muscles of the larynx, pharynx, and esophagus to regulate the motor function of speech and swallowing. To identify candidate cardiovagal neurons and differentiate them from neighboring motor neurons, we profiled genome-wide RNA expression in ~1,250 nAmb neurons by single nuclei RNA-sequencing. Clustering these neurons based on their transcriptomic similarity revealed six candidate molecular subtypes, which we annotated according to their subtype-specific marker genes. Based on their gene expression profiles, we hypothesized that three subtypes may be cardiovagal neurons. Three other subtypes, marked by Vipr2 or Crhr2 expression, likely correspond to the neurons that innervate the upper airways and esophagus, as shown by our previous work. Two of the three potential cardiovagal subtypes were specifically marked by their expression of Vip and Adcyap1, respectively, and the other subtype co-expressed Npy2r and Adcyap1. Fluorescence RNA in situ hybridization validated that Npy2r and Adcyap1 transcripts mark partially overlapping nAmb neurons residing throughout its rostrocaudal axis. To determine whether these neuron subtypes can control heart rate, we targeted expression of a calcium-translocating channelrhodopsin variant (CatCh) to the Npy2r+ and Adcyap1+ nAmb neuron subtypes using intersectional genetics. We then activated CatCh+ neuron subtypes using laser light transmitted via a fiber optic implanted over the nAmb and measured the effect on heart rate (HR) in awake, behaving mice. Our results show an immediate and dramatic decrease in HR upon activation of Npy2r+nAmb neurons (n=6 Npy2r-Cre:Chat-Flp:CatCh mice; laser-on HR= 58 ± 17% of laser-off HR; p&lt;0.0001), or Adcyap1+nAmb neurons (n=5 Adcyap1-Cre:Chat-Flp:CatCh mice; laser-on HR= 34 ± 13% of laser-off HR; p&lt;0.0001). The HR decrease depended on the frequency of stimulation (n=3 mice per genotype) and was not observed in the absence of CatCh expression (n=4 mice; laser-on HR = 100 ± 0% of laser-off HR; p&gt;0.05). Blocking muscarinic receptors with atropine prevented the bradycardic effect of activating Npy2r+nAmb neurons (n=5 mice), suggesting a parasympathetic mechanism. These results indicate that Npy2r+nAmb neurons and Adcyap1+nAmb neurons are capable of suppressing HR, which builds on our previous observation of bradycardia when activating all nAmb neurons but not when activating the upper airway and esophageal projecting subtypes (recently published) Vipr2+nAmb neurons and Crhr2+nAmb neurons, respectively. Together, our results identify cardiovagal neuron subtypes in the nAmb based on their molecular and functional properties. The authors gratefully acknowledge Maisie Crook for her technical assistance. Funding was provided by National Institutes for Health (NIH) F31 HL158187 to T.C.C.; NIH R01 HL148004 to S.B.G.A.; and an American Diabetes Association Pathway to Stop Diabetes Initiator Award 1-18-INI-14 and NIH R01 HL153916 to J.N.C. This is the full abstract presented at the American Physiology Summit 2023 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.

Disambiguating control of the diving reflex by the nucleus ambiguus

Heart rate at rest and during cardiac reflexes is set primarily by cardiovagal neurons (CVNs) in the nucleus ambiguus (nAmb), which innervate the heart via the vagus nerve. These CVNs are thought to control the diving reflex, a powerful and highly conserved mammalian autonomic reflex that robustly decreases heart rate during voluntary underwater diving to preserve oxygen levels in the heart and brain. However, the identity of the CVNs mediating diving reflex bradycardia remains unknown. Previous studies in our lab uncovered three molecularly distinct subtypes of nAmb neurons, one of which innervates the heart, is capable of robustly decreasing heart rate, and is marked by its expression of the gene Npy2r (Npy2rnAmb). Accordingly, we hypothesize that Npy2rnAmb neurons are responsible for the decrease in heart rate during the diving reflex. To test this hypothesis, we first asked whether Npy2rnAmb neurons are activated by voluntary diving. Over the course of 6 days, mice (n=17) were trained to voluntarily dive about 0.5 meters underwater in a custom-built tank, with each dive lasting about 2 seconds. Upon completion of training, mice were injected intraperitoneally with 0.05 mL of the retrograde tracer Fluorogold to fluorescently label all nAmb neurons. On the day of testing, the experimental group of dive-trained mice (n=9) underwent 15 minutes of consecutive voluntary dives, about 40 dives each. After diving, mice recovered in a warm cage for 30-40 minutes and were then anesthetized and transcardially perfused with fixative. For controls, the same procedure was performed on another group of dive-trained mice (n=8), except that the dive chamber only contained enough water to wet their paws (no diving occurred). After tissue fixation, the medulla was extracted, coronally sectioned, and processed for RNA fluorescence in situ hybridization (RNA FISH) to visualize transcripts for Npy2r and the immediate early gene, Fos. There was a significant increase in the proportion of Fluorogold+ Npy2r+ nAmb neurons that expressed Fos after diving compared to controls (45 ± 6% vs. 16 ± 4% triple positive, n=7 &amp; 6, P=0.0047, Mann Whitney test), indicating that Npy2rnAmb neurons are activated during voluntary diving. These data raise the possibility that Npy2rnAmb neurons are CVNs that mediate the diving reflex. The authors gratefully acknowledge Maisie Crook for her technical assistance throughout this project. Funding was provided by the UVA Biology Schwager Summer Research Scholarship and UVA Harrison Undergraduate Research Award to V.G.; National Institutes for Health (NIH) F31 HL158187 to T.C.C.; NIH R01 HL148004 to S.B.G.A.; and an American Diabetes Association Pathway to Stop Diabetes Initiator Award 1-18-INI-14 and NIH R01 HL153916 to J.N.C. This is the full abstract presented at the American Physiology Summit 2023 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.

Unpacking the multimodal, multi-scale data of the fast and slow lanes of the cardiac vagus through computational modelling.

What is the topic of this review? The vagus nerve is a crucial regulator of cardiovascular homeostasis, and its activity is linked to heart health. Vagal activity originates from two brainstem nuclei: the nucleus ambiguus (fast lane) and the dorsal motor nucleus of the vagus (slow lane), nicknamed for the time scales that they require to transmit signals. What advances does it highlight? Computational models are powerful tools for organizing multi-scale, multimodal data on the fast and slow lanes in a physiologically meaningful way. A strategy is laid out for how these models can guide experiments aimed at harnessing the cardiovascular health benefits of differential activation of the fast and slow lanes. The vagus nerve is a key mediator of brain-heart signaling, and its activity is necessary for cardiovascular health. Vagal outflow stems from the nucleus ambiguus, responsible primarily for fast, beat-to-beat regulation of heart rate and rhythm, and the dorsal motor nucleus of the vagus, responsible primarily for slow regulation of ventricular contractility. Due to the high-dimensional and multimodal nature of the anatomical, molecular and physiological data on neural regulation of cardiac function, data-derived mechanistic insights have proven elusive. Elucidating insights has been complicated further by the broad distribution of the data across heart, brain and peripheral nervous system circuits. Here we lay out an integrative framework based on computational modelling for combining these disparate and multi-scale data on the two vagal control lanes of the cardiovascular system. Newly available molecular-scale data, particularly single-cell transcriptomic analyses, have augmented our understanding of the heterogeneous neuronal states underlying vagally mediated fast and slow regulation of cardiac physiology. Cellular-scale computational models built from these data sets represent building blocks that can be combined using anatomical and neural circuit connectivity, neuronal electrophysiology, and organ/organismal-scale physiology data to create multi-system, multi-scale models that enable in silico exploration of the fast versus slow lane vagal stimulation. The insights from the computational modelling and analyses will guide new experimental questions on the mechanisms regulating the fast and slow lanes of the cardiac vagus toward exploiting targeted vagal neuromodulatory activity to promote cardiovascular health.

Early central cardiovagal dysfunction after high fat diet in a murine model

High fat diet (HFD) promotes cardiovascular disease and blunted cardiac vagal regulation. Temporal onset of loss of cardiac vagal control and its underlying mechanism are presently unclear. We tested our hypothesis that reduced central vagal regulation occurs early after HFD and contributes to poor cardiac regulation using cardiovascular testing paired with pharmacology in mice, molecular biology, and a novel bi-transgenic mouse line. Results show HFD, compared to normal fat diet (NFD), significantly blunted cardio/pulmonary chemoreflex bradycardic responses after 15 days, extending as far as tested (> 30 days). HFD produced resting tachycardia by day 3, reflected significant loss of parasympathetic tone. No differences in bradycardic responses to graded electrical stimulation of the distal cut end of the cervical vagus indicated diet-induced differences in vagal activity were centrally mediated. In nucleus ambiguus (NA), surface expression of δ-subunit containing type A gamma-aminobutyric acid receptors (GABAA(δ)R) increased at day 15 of HFD. Novel mice lacking δ-subunit expression in vagal motor neurons (ChAT-δnull) failed to exhibit blunted reflex bradycardia or resting tachycardia after two weeks of HFD. Thus, reduced parasympathetic output contributes to early HFD-induced HR dysregulation, likely through increased GABAA(δ)Rs. Results underscore need for research on mechanisms of early onset increases in GABAA(δ)R expression and parasympathetic dysfunction after HFD.

Subject-Specific Activation of Central Respiratory Centers during Breath-Holding Functional Magnetic Resonance Imaging

Background: Voluntary breath-holding (BH) triggers responses from central neural control and respiratory centers in order to restore breathing. Such responses can be observed using functional MRI (fMRI). Objectives: We used this paradigm in healthy volunteers with the view to develop a biomarker that could be used to investigate disorders of the central control of breathing at the individual patient level. Method: In 21 healthy human subjects (mean age±SD, 32.8 ± 9.9 years old), fMRI was used to determine, at both the individual and group levels, the physiological neural response to expiratory and inspiratory voluntary apneas, within respiratory control centers in the brain and brainstem. Results: Group analysis showed that expiratory BH, but not inspiratory BH, triggered activation of the pontine respiratory group and raphe nuclei at the group level, with a significant relationship between the levels of activation and drop in SpO2. Using predefined ROIs, expiratory BH, and to a lesser extent, inspiratory BH were associated with activation of most respiratory centers. The right ventrolateral nucleus of the thalamus, right pre-Bötzinger complex, right VRG, right nucleus ambiguus, and left Kölliker-Fuse-parabrachial complex were only activated during inspiratory BH. Individual analysis identified activations of cortical/subcortical and brainstem structures related to respiratory control in 19 out of 21 subjects. Conclusion: Our study shows that BH paradigm allows to reliably trigger fMRI response from brainstem and cortical areas involved in respiratory control at the individual level, suggesting that it might serve as a clinically relevant biomarker to investigate conditions associated with an altered central control of respiration.

Restoring the Autonomic Balance in an Atrial Fibrillation Rat Model by Electroacupuncture at the Neiguan Point

ObjectivesAutonomic nervous activity imbalance plays an important role in atrial fibrillation (AF). AF can be treated by acupuncture at the Neiguan point (PC6), but the mechanism remains elusive. Here, we investigated autonomic nervous system activity in electroacupuncture (EA) at PC6 in a rat AF model. Material and MethodsIn this study, we established a rat AF model via tail vein injection with ACh-CaCl2 for ten consecutive days with or without EA at PC6. AF inducibility and heart rate variability (HRV) were assessed by electrocardiogram. Next, we completed in vivo recording of the activity of cervical sympathetic and vagal nerves, respectively. Finally, the activities of brain regions related to autonomic nerve regulation were assessed by c-Fos immunofluorescence and multichannel recording. ResultsEA at PC6 decreased AF inducibility and prevented changes in HRV caused by ACh-CaCl2 injection. Meanwhile, EA at PC6 reversed the increased sympathetic and decreased vagal nerve activity in AF rats. Furthermore, EA treatment downregulated increased c-Fos expression in brain regions, including paraventricular nucleus, rostral ventrolateral medulla, and dorsal motor nucleus of the vagus in AF, while c-Fos expression in nucleus ambiguus was upregulated with EA. ConclusionThe protective effect of EA at PC6 on AF is associated with balance between sympathetic and vagal nerve activities.

The autonomic nervous system: A potential link to the efficacy of acupuncture.

The autonomic nervous system (ANS) is a diffuse network that regulates physiological systems to maintain body homeostasis by integrating inputs from the internal and external environment, including the sympathetic, parasympathetic, and enteric nervous systems (ENS). Recent evidence suggests that ANS is one of the key neural pathways for acupuncture signal transduction, which has attracted worldwide attention in the acupuncture field. Here, we reviewed the basic and clinical research published in PubMed over the past 20 years on the effects of acupuncture on ANS regulation and homeostasis maintenance. It was found that acupuncture effectively alleviates ANS dysfunction-associated symptoms in its indications, such as migraine, depression, insomnia, functional dyspepsia, functional constipation. Acupuncture stimulation on some specific acupoints activates sensory nerve fibers, the spinal cord, and the brain. Using information integration and efferents from a complex network of autonomic nuclei of the brain, such as the insular cortex (IC), prefrontal cortex, anterior cingulate cortex (ACC), amygdala (AMG), hypothalamus, periaqueductal gray (PAG), nucleus tractus solitarius (NTS), ventrolateral medulla (VLM), nucleus ambiguus (AMB), acupuncture alleviates visceral dysfunction, inflammation via efferent autonomic nerves, and relieves pain and pain affect. The modulating pattern of sympathetic and parasympathetic nerves is associated with acupuncture stimulation on specific acupoints, intervention parameters, and disease models, and the relationships among them require further exploration. In conclusion, ANS is one of the therapeutic targets for acupuncture and mediates acupuncture's actions, which restores homeostasis. A systemic study is needed to determine the rules and mechanisms underlying the effects of acupoint stimulation on corresponding organs mediated by specific central nervous networks and the efferent ANS.

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

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. Laboratory experiment. 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. 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. 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. NA Laryngoscope, 133:2240-2247, 2023.

Multiple cranial palsy in pasein with cerebral cavernoma malformations on the brain stem

Facial palsy is a condition that is often encountered in neurological practice. The weakness of closing the eyes can be found in cases of central facial paralysis. Facial palsy is characterized by acute, unilateral, partial, or complete facial paralysis. Cerebral cavernous malformation is one rare form of lesion, accounting for about 5 to 13% of blood vessel anomalies in the central nervous system. Brain stem cavernomas are rare and less than 20% intracerebral cavernomas. Most patients with cerebral cavernoma of the brain stem have symptoms of acute neurological deficits. The inferior cerebellar pedunkel, vestibular nucleus, trigeminal nucleus of the spine, and ambiguous nucleus are usually affected. Clinical signs include vestibulocerebellar symptoms such as vertigo, falling to the side of lesions, diplopia, multidirectional nystagmus, ipsilateral Horner syndrome, hiccups, contralateral body weakness, pain and sensation of temperature, motion, dysphonia, dysphagia, dysarthria, and decreased gag reflex (Sciacca et al., 2019). A 26-year-old man with a right eye cannot close, the right face cannot be moved since 4 months, the right side face feels thick from the right cheek to the right chin. The patient has no previous history of stroke. In the examination obtained hipestesi NV2 and NV3 right, facial palsy peripheral right.

When laughter arrests speech: fMRI-based evidence.

Who has not experienced that sensation of losing the power of speech owing to an involuntary bout of laughter? An investigation of this phenomenon affords an insight into the neuronal processes that underlie laughter. In our functional magnetic resonance imaging study, participants were made to laugh by tickling in a first condition; in a second one they were requested to produce vocal utterances under the provocation of laughter by tickling. This investigation reveals increased neuronal activity in the sensorimotor cortex, the anterior cingulate gyrus, the insula, the nucleus accumbens, the hypothalamus and the periaqueductal grey for both conditions, thereby replicating the results of previous studies on ticklish laughter. However, further analysis indicates the activity in the emotion-associated regions to be lower when tickling is accompanied by voluntary vocalization. Here, a typical pattern of activation is identified, including the primary sensory cortex, a ventral area of the anterior insula and the ventral tegmental field, to which belongs to the nucleus ambiguus, namely, the common effector organ for voluntary and involuntary vocalizations. During the conflictual voluntary-vocalization versus laughter experience, the laughter-triggering network appears to rely heavily on a sensory and a deep interoceptive analysis, as well as on motor effectors in the brainstem.This article is part of the theme issue ‘Cracking the laugh code: laughter through the lens of biology, psychology and neuroscience’.