Vagal Neurons Research Articles

KHz-frequency electrical stimulation selectively activates small, unmyelinated vagus afferents

BackgroundVagal reflexes regulate homeostasis in visceral organs and systems through afferent and efferent neurons and nerve fibers. Small, unmyelinated, C-type afferents comprise over 80% of fibers in the vagus and form the sensory arc of autonomic reflexes of the gut, lungs, heart and vessels and the immune system. Selective bioelectronic activation of C-afferents could be used to mechanistically study and treat diseases of peripheral organs in which vagal reflexes are involved, but it has not been achieved. MethodsWe stimulated the vagus in rats and mice using trains of kHz-frequency stimuli. Stimulation effects were assessed using neuronal c-Fos expression, physiological and nerve fiber responses, optogenetic and computational methods. ResultsIntermittent kHz stimulation for 30 min activates specific motor and, preferentially, sensory vagus neurons in the brainstem. At sufficiently high frequencies (>5 kHz) and at intensities within a specific range (7–10 times activation threshold, T, in rats; 15-25 × T in mice), C-afferents are activated, whereas larger, A- and B-fibers, are blocked. This was determined by measuring fiber-specific acute physiological responses to kHz stimulus trains, and by assessing fiber excitability around kHz stimulus trains through compound action potentials evoked by probing pulses. Aspects of selective activation of C-afferents are explained in computational models of nerve fibers by how fiber size and myelin shape the response of sodium channels to kHz-frequency stimuli. ConclusionkHz stimulation is a neuromodulation strategy to robustly and selectively activate vagal C-afferents implicated in physiological homeostasis and disease, over larger vagal fibers.

SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping

SummarySpatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.

Infectious and Inflammatory Pathways to Cough.

Coughing is a dynamic physiological process resulting from input of vagal sensory neurons innervating the airways and perceived airway irritation. Although cough serves to protect and clear the airways, it can also be exploited by respiratory pathogens to facilitate disease transmission. Microbial components or infection-induced inflammatory mediators can directly interact with sensory nerve receptors to induce a cough response. Analysis of cough-generated aerosols and transmission studies have further demonstrated how infectious disease is spread through coughing. This review summarizes the neurophysiology of cough, cough induction by respiratory pathogens and inflammation, and cough-mediated disease transmission.

Gut–brain circuits for fat preference

The perception of fat evokes strong appetitive and consummatory responses1. Here we show that fat stimuli can induce behavioural attraction even in the absence of a functional taste system2,3. We demonstrate that fat acts after ingestion via the gut–brain axis to drive preference for fat. Using single-cell data, we identified the vagal neurons responding to intestinal delivery of fat, and showed that genetic silencing of this gut-to-brain circuit abolished the development of fat preference. Next, we compared the gut-to-brain pathways driving preference for fat versus sugar4, and uncovered two parallel systems, one functioning as a general sensor of essential nutrients, responding to intestinal stimulation with sugar, fat and amino acids, whereas the other is activated only by fat stimuli. Finally, we engineered mice lacking candidate receptors to detect the presence of intestinal fat, and validated their role as the mediators of gut-to-brain fat-evoked responses. Together, these findings reveal distinct cells and receptors that use the gut–brain axis as a fundamental conduit for the development of fat preference.

Pulmonary neuroendocrine cells sense succinate to stimulate myoepithelial cell contraction.

Pulmonary neuroendocrine cells (PNECs) are rare airway cells with potential sensory capacity linked to vagal neurons and immune cells. How PNECs sense and respond to external stimuli remains poorly understood. We discovered PNECs located within pig and human submucosal glands, a tissue that produces much of the mucus that defends the lung. These PNECs sense succinate, an inflammatory molecule in liquid lining the airway surface. The results indicate that succinate migrates down the submucosal gland duct to the acinus, where it triggers apical succinate receptors, causing PNECs to release ATP. The short-range ATP signal stimulates the contraction of myoepithelial cells wrapped tightly around the submucosal glands. Succinate-triggered gland contraction may complement the action of neurotransmitters that induce mucus release but not gland contraction to promote mucus ejection onto the airway surface. These findings identify a local circuit in which rare PNECs within submucosal glands sense an environmental cue to orchestrate the function of airway glands.

Structure and Functions of the Vagus Nerve in Mammals.

We review the structure and function of the vagus nerve, drawing on information obtained in humans and experimental animals. The vagus nerve is the largest and longest cranial nerve, supplying structures in the neck, thorax, and abdomen. It is also the only cranial nerve in which the vast majority of its innervation territory resides outside the head. While belonging to the parasympathetic division of the autonomic nervous system, the nerve is primarily sensory-it is dominated by sensory axons. We discuss the macroscopic and microscopic features of the nerve, including a detailed description of its extensive territory. Histochemical and genetic profiles of afferent and efferent axons are also detailed, as are the central nuclei involved in the processing of sensory information conveyed by the vagus nerve and the generation of motor (including parasympathetic) outflow via the vagus nerve. We provide a comprehensive review of the physiological roles of vagal sensory and motor neurons in control of the cardiovascular, respiratory, and gastrointestinal systems, and finish with a discussion on the interactions between the vagus nerve and the immune system. © 2022 American Physiological Society. Compr Physiol 12: 1-49, 2022.

The neural basis of sugar preference.

When it comes to food, one tempting substance is sugar. Although sweetness is detected by the tongue, the desire to consume sugar arises from the gut. Even when sweet taste is impaired, animals can distinguish sugars from non-nutritive sweeteners guided by sensory cues arising from the gut epithelium. Here, we review the molecular receptors, cells, circuits and behavioural consequences associated with sugar sensing in the gut. Recent work demonstrates that some duodenal cells, termed neuropod cells, can detect glucose using sodium-glucose co-transporter 1 and release glutamate onto vagal afferent neurons. Based on these and other data, we propose a model in which specific populations of vagal neurons relay these sensory cues to distinct sets of neurons in the brain, including neurons in the caudal nucleus of the solitary tract, dopaminergic reward circuits in the basal ganglia and homeostatic feeding circuits in the hypothalamus, that alter current and future sugar consumption. This emerging model highlights the critical role of the gut in sensing the chemical properties of ingested nutrients to guide appetitive decisions.

Molecular cloning of cholecystokinin (CCK) and CCK-A receptor and mechanism of CCK-induced gastrointestinal motility in Suncus murinus

Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.

Thyrotropin-releasing hormone induces Ca2+ increase in a subset of vagal nodose ganglion neurons

Thyrotropin-releasing hormone (TRH) plays a central role in metabolic homeostasis, and single-cell sequencing has recently demonstrated that vagal sensory neurons in the nodose ganglion express thyrotropin-releasing hormone receptor 1 (TRHR1). Here, in situ hybridization validated the presence of TRHR1 in nodose ganglion (NG) neurons and immunohistochemistry showed that the receptor is expressed at the protein level. However, it has yet to be demonstrated whether TRHR1 is functionally active in NG neurons. Using NG explants transduced with a genetically encoded Ca2+ indicator (GECI), we show that TRH increases Ca2+ in a subset of NG neurons. TRH-induced Ca2+ transients were briefer compared to those induced by CCK-8, 2-Me-5-HT and ATP. Blocking Na+ channels with TTX or Na+ substitution did not affect the TRH-induced Ca2+ increase, but blocking Gq signaling with YM-254890 abolished the TRH-induced response. Field potential recordings from the vagus nerve in vitro showed an increase in response to TRH, suggesting that TRH signaling produces action potentials in NG neurons. These observations indicate that TRH activates a small group of NG neurons, involving Gq pathways, and we hypothesize that these neurons may play a role in gut-brain signaling.

1315-P: Optogenetic Activation of Vagal Motor Neurons in Mice Modulates Heart Rate but Not Blood Glucose Levels

Parasympathetic neurons in the dorsal motor nucleus of the vagus (DMV) control glucose metabolism through the vagus nerve and so are potential targets for diabetes treatment. Yet, little is known about the molecular identity and organization of glucose-regulating DMV neurons. To identify these neurons for further investigation, we optogenetically activated them in mice while measuring blood glucose. We targeted a light-sensitive ion channel, CaTCh, selectively to DMV neurons based on their co-expression of Chat and Phox2b, which resulted in CaTCh expression in most or all DMV neurons but no surrounding neurons. To photostimulate CaTCh+ DMV neurons, we then implanted optical fibers over the DMV in 9 adult Chat::Phox2b::CaTCh mice (4 females, 5 males; mean age ± S.D., 24 ± 13 weeks) . After acclimation we fasted the mice overnight and then delivered pulses of blue laser light (20Hz, 10ms pulse, 3 sec off, 1 sec on) for 15 min to activate DMV neurons. We used a glucometer on tail blood samples drawn every 5 minutes for 1 hour, beginning 20 minutes before and ending 25 minutes after photostimulation. Before, during, and after photostimulation, blood glucose averaged 2± 35 mg/dL, 220 ± 36 mg/dL, and 215 ± 45 mg/dL and did not change significantly with photostimulation (p>0.05, ANOVA) . To rule out stress as a confound, we repeated these studies after anesthetizing 4-hour fasted mice with isoflurane but still did not detect a significant effect of photostimulation on blood glucose. Interestingly, however, heart rate decreased significantly with photostimulation (pre, 5± 23 bpm; during, 429 ± 32 bpm; post, 517 ± 24 bpm; p<0.001, ANOVA) , indicating that the photostimulation parameters were sufficient to activate vagal neurons. Further work is underway to address whether the lack of effect of DMV poststimulation on blood glucose is due to antagonistic signaling (e.g., coincident release of insulin and glucagon) or represents a limitation of the method. Disclosure J.Campbell: None. N.J.Conley: None. L.S.Kauffman: None. Funding American Diabetes Association (1-18-INI-14) ;

Bile acids induce Ca2+ signaling and membrane permeabilizations in vagal nodose ganglion neurons

Bile acids (BAs) play an important role in the digestion of dietary fats and act as signaling molecules. However, due to their solubilizing properties, high concentrations in the gut may negatively affect gut epithelium and possibly afferent fibers innervating the gastrointestinal tract (GI). To determine the effect of BAs on intracellular Ca2+ and membrane permeabilization we tested a range of concentrations of two BAs on vagal nodose ganglion (NG) neurons, Chinese Hamster Ovary (CHO), and PC12 cell lines. NG explants from mice were drop-transduced with the genetically encoded Ca2+ indicator AAV9-Syn-jGCaMP7s and used to measure Ca2+ changes upon application of deoxycholic acid (DCA) and taurocholic acid (TCA). We found that both BAs induced a Ca2+ increase in NG neurons in a dose-dependent manner. The DCA-induced Ca2+ increase was dependent on intracellular Ca2+ stores. NG explants, with an intact peripheral part of the vagus nerve, showed excitation of NG neurons in nerve field recordings upon exposure to DCA. The viability of NG neurons at different BA concentrations was determined, and compared to CHO and PC12 cells lines using propidium iodide labeling, showing threshold concentrations of BA-induced cell death at 400–500 μM. These observations suggest that BAs act as Ca2+-inducing signaling molecules in vagal sensory neurons at low concentrations, but induce cell death at higher concentrations, which may occur during inflammatory bowel diseases.

Vagus Nerve Sensory Neurons Respond Distinctly to Specific Inflammatory Mediators

The peripheral nervous system communicates with the immune system in response to injury or infection. In the case of inflammation, sensory signals travel up to the brain via the vagus nerve, which is a major pathway for neuro‐immune communication. While previous work from our group showed that cytokines administered systemically were associated with specific patterns of vagus nerve activity, we did not know how individual vagal sensory neurons encoded this immune information.Here, we use in vivo calcium imaging to monitor the neural activity of individual vagal sensory neurons in response to specific inflammatory mediators. Using mice that express the calcium sensor GCaMP6f in glutamatergic neurons, we imaged neurons of the nodose ganglion in situ using a 1‐photon miniature microscope (Miniscope). During imaging, the inflammatory mediators tumor necrosis factor (TNF) and interleukin 1β (IL‐1β) were applied directly to the vagus nerve. The raw fluorescence data was analyzed with a Python‐based calcium imaging analysis (CaImAn) software and a customized pipelines to output the neural activity (change in fluorescence: ∆F) of individually detected neurons. An average of 69±16.3 neurons were recorded per 1 hour imaging session.Our results reveal that specific vagal sensory neurons respond to differentially to specific immune mediators. The average amplitude, integral, and delay of TNF‐responsive neurons was significantly higher than IL‐1β‐responsive cells (TNF vs IL‐1β, p < 0.01). This result suggests that TNF application triggers responses later and with higher firing rates in responsive neurons compared to IL‐1β. Neuronal responses to IL‐1β had a significantly higher number of peaks when compared to TNF responses (IL‐1β vs TNF, p < 0.05). When compared to responses for the TRPV1‐specific agonist capsaicin, the cytokine responses were found to have significantly lower number of peaks (capsaicin vs IL‐1β, p < 0.05), which suggests that immune mediators produce different neural activity than canonical activators. Taken together, our findings demonstrate that individual vagal sensory neurons encode information about distinct inflammatory stimuli and provide insight into the neural sensing of inflammation. Further investigation into this type of neuro‐immune signaling may identify novel neural targets for the treatment of inflammatory disorders.

Asymmetric Control of Food Intake by Left and Right Vagal Sensory Neurons

Gut‐innervating vagal sensory neurons residing in the nodose ganglia (NG) conveys meal‐borne signals and control food intake, however, the underlying gut‐brain neurocircuit is unclear. The neuropeptide cocaine‐ and amphetamine‐regulated transcript (CART) is expressed in a subpopulation of vagal sensory neurons (~40%) that innervate the whole length of the gut and its release in the NTS is sufficient for meal termination. Thus, it can be an excellent target to understand the role of sensory neurons in feeding. Emerging evidence suggests that right or left NG (LNG or RNG) neurons recruit different downstream central circuits to control feeding, but the mechanisms remain poorly understood. Our hypothesis is that LNGCART and RNGCART neurons sense different meal‐related signals and are necessary and sufficient to modulate feeding.MethodsIn vivo calcium imaging, viral neuronal tracing, and feeding behaviors were assessed in response to chemo‐ and opto‐genetic stimulation of gut‐innervating vagal sensory neurons using CARTcre mice.ResultsAnatomical tracing revealed that LNGCART and RNGCART neurons innervate different regions of the duodenum (n=3). LNGCART innervate the muscular layer, while RNGCART innervate the villi. Importantly, 2‐photon calcium imaging highlighted real time increased activity in LNGCART in response to stretch, while intraduodenal fat infusions preferentially increased RNGCART neuron activity (n=7/group). Mice with LNGCART neurons ablation consumed higher volumes of liquid meal indicative of attenuated response to gastric and intestinal distension (n=6‐7/group). Furthermore, chemogenetic stimulation of LNGCART, but not RNGCART, caused pronounced and sustained hypophagia (n=7/group). Interestingly, mice with chemogenetic activation of RNGCART, but not LNGCART, increased flavor conditioning (n=7/group), while ablation of RNGCART neurons abolished the conditioned flavor preference that was paired with intragastric fat infusion (n=7/group) supporting their role in reward. In addition, optogenetic stimulation of RNGCART promoted self‐stimulation in a nose poke task (n=4). Lastly, dopaminergic neurons of the substantia nigra increased their firing rate in response to intraduodenal fat infusion, but not duodenal distension (n=2/group).ConclusionLeft and right vagus nerves provide different sensory information about a meal to the brain. Both anatomical and behavioral feeding studies support the necessity and sufficiency LNGCART neurons as food quantity detectors and RNGCART neurons as food reward mediators ‐ which has less influence on how much is consumed but rather plays a role in the decision‐making process about which foods to consume. Our findings on asymmetrical vagal innervation of the gut provides new mechanistic insight for how feeding decisions are made in response to vast and complex meal‐related signals.

GABAA δ receptors in vagal motor neurons play a role in the influence of high fat diet on cardiac function

Central vagal circuitry plays a key role in cardioinhibition and is critical to cardiac homeostasis. Long‐term high fat diet (HFD) is associated with decreased vagal drive, which is linked to increased cardiovascular morbidity and mortality. However, recent evidence suggests that HFD elicits changes in central circuits during early consumption (days). This study tested the hypothesis that decreased central vagal regulation contributes to poor cardiac regulation during the first two weeks of HFD. C57/Bl6 mice were examined for changes in autonomic regulation of HR both at rest and during reflex challenges using chronically implanted telemetry devices. Metabolic parameters were examined throughout all experiments. HFD fed mice exhibited a resting tachycardia throughout the two weeks of HFD (669 ± 20 bpm) compared to NFD (606 ± 20 bpm; p=0.03). Using pharmacological manipulations at Day 15, animals given HFD showed lower resting HR responses to intraperitoneal injection of the parasympathetic antagonist, methyl‐scopolamine, (68 ± 8 bpm) compared to NFD (104 ± 15 bpm; p=0.03). To determine if HFD also influenced homeostatic regulation of HR, animals were tested for capsaicin‐ and phenylbiguanide (PBG)‐mediated cardiopulmonary reflexes. In these animals, HFD blunted capsaicin‐mediated cardiopulmonary reflex at day 15 of HFD (‐19 ± 7 bpm), compared to NFD (‐45 ± 3 bpm; p=0.01). The HFD‐induced blunting of capsaicin‐mediated bradycardia continued throughout testing (60 days; ‐22 ± 5 bpm in HFD). No changes were seen in PBG‐mediated bradycardias (p=0.86). Previous work from our lab indicates that vagal motor neurons after metabolic challenges express large GABAergic currents mediated by GABAA receptors expressing the δ‐subunit, GABAA(δ)R. Since GABAergic neurotransmission is critical to both resting HR and reflex responses, we generated a novel bi‐transgenic mouse line using a floxed GABAA(δ)R and ChATcre mouse line (ChAT‐ δnull) to knock out the expression of δ‐subunit in vagal motor neurons. Examination of resting HR after HFD and NFD in ChAT‐ δnull showed no differences between diets (p=0.68). Pharmacological manipulations of HR in ChAT‐δnull mice with methyl‐scopolamine also showed no differences in HR responses between the diets (p=0.34). In addition, ChAT‐ δnull mice on HFD (61 ± 9 bpm) no longer showed a significant blunting of capsaicin‐mediated bradycardia compared to NFD (39 ± 5 bpm; p=0.10). Together, this study showed that HFD induces lasting changes in central circuitry within 15 days after consumption. In addition, this study found that parasympathetic dysfunction contributes to early HFD‐induced changes in HR and autonomic reflexes. This is important, as most pharmaceutical treatments currently available to patients target the sympathetic hyperactivity, and not parasympathetic hypoactivity. Finally, increased GABAA(δ)Rs likely mediate decreased vagal output after HFD. Therefore, more research is needed to determine mechanistic underpinning(s) of increased GABAA(δ)R activity and parasympathetic dysfunction to lead to better treatment options for cardiovascular disease associated with high fat diet consumption.

High resolution retrograde tracing, morphological analysis, RNA sequencing and differential expression analysis of identified vagal afferent neurons innervating the muscle or mucosa in 3 different stomach regions in the rat

The functions of the stomach are powerfully influenced by the vagus nerve and vago‐vagal reflexes. The vagal afferent neurons that innervate the stomach and monitor its functional state have clearly demonstrated roles in appetite regulation, gastric accommodation and gastric emptying. In mammals with single compartment stomachs, including humans, the stomach can be divided into functional regions and the muscle and mucosal layers show region‐specific specializations. Electrophysiological and morphological studies suggest that the vagal sensory axons innervating the different regions and layers of the stomach wall have distinct properties and participate in different types of reflex controls. We therefore hypothesized that these functional and structural differences would be reflected in distinct gene expression profiles of afferent neurons, depending on the stomach region and tissue innervated. We developed methods to selectively retrogradely label afferent neurons innervating the muscle or mucosa in the corpus and antrum and the muscle in the fundus in the rat, using local microinjections of fluorescent nanospheres that diffuse minimally. Up to 30 microinjections of 30‐100nL of tracer beads were made into either the muscle or the mucosa in one stomach region in each rat under anesthesia. The animal was allowed to recover and kept for 1‐2 weeks before being humanely killed and tissues harvested. Labelled neurons were studied in situ in the nodose ganglion using confocal microscopy, immunohistochemistry and fluorescence in situ hybridization, and individual neurons collected after dissociation for RNAseq analysis. RNA sequencing was performed using pooled samples of 5‐12 neurons labelled from each region and deep sequencing (30‐50 million reads per sample), and then analyzed using minimal filtering to allow detection of genes expressed at low copy number.Nodose neurons labelled from the mucosa were significantly larger than those labelled from the muscle in both the corpus and antrum, whether quantified in situ or after dissociation, confirming that microinjections into the different stomach wall layers labelled different populations of neurons. Pairwise differential expression analyses of RNAseq data showed distinct mRNA expression profiles depending on the region innervated, and between muscle and mucosal innervating neurons in each region, based on Benjamini/Hochberg adjusted P values (Limma analysis tool). Some genes displayed region specific expression patterns while others showed tissue specific patterns (eg muscle vs mucosa) and some showed both. Among differentially expressed genes were some previously identified to specifically label gastric mucosal and muscle afferent neurons, such as somatostatin, CCKB receptor and GRP65. In addition, many other genes were differentially expressed and mRNA for genes known to be functional in vagal afferent neurons, such as ghrelin, leptin and galanin receptors, were detected. Differential expression patterns for various genes were confirmed in labelled neurons in situ using immunohistochemistry and/or in situhybridization. The data provide a rich resource for further studies of the visceral sensation in the different stomach regions and how region‐specific signaling might regulate appetite and gastric function in both normal and disease states.

Vagus nerve sensory neurons have distinct neural responses to inflammatory mediators

Abstract Cytokines are secreted signaling proteins that are important mediators of inflammation. While prior work has demonstrated that the level of cytokines can be regulated by nerve stimulation, the role of the nervous system in sensing these immune mediators is still poorly understood. During periods of inflammation, it has been shown that sensory signals travel up the vagus nerve to the brain. However, it is unclear how individual vagal sensory neurons encode specific immune information. Here we use in vivo calcium imaging of the nodose ganglion to monitor neural activity in individual vagus nerve sensory neurons in response to specific cytokines. Using mice that express the calcium indicator GCaMP6f in glutamatergic neurons, we imaged neurons of the nodose ganglion in situ using a 1-photon miniature microscope (Miniscope). During imaging, the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor (TNF) were applied directly to the vagus nerve. Fluorescence data was analyzed with a Python-based software CaImAn and a custom pipeline to output the single neuron activity as change in fluorescence: ΔF/F. Our results reveal that specific vagal sensory neurons respond differentially to specific immune mediators. The average amplitude, integral, and delay of TNF-responsive neurons was significantly higher than IL-1β-responsive cells (TNF vs IL-1β, p < 0.01), while having less number of peaks per response (TNF vs IL-1β, p < 0.05). This may suggest different patterns of transient neural activity associated with each particular cytokine. Further investigation into this type of neuro-immune signaling may identify novel neural targets for the treatment of inflammatory disorders. This study was funded in part by NIH/NIGMS

Excitation of Vagal Afferent Neurons by Fecal Supernatant from Inflammatory Bowel Disease Patients

BackgroundThe gut‐ microbiota‐brain axis has received increasing attention recently due to evidence that colonic microbes can affect brain function and behavior. Recent studies have demonstrated that vagal afferent neurons may be an important conduit between the gut microbiota and the brain, as it is capable of detecting mediators released from the gut microbiota. However, it is unknown whether i) the gut microbiota from healthy human stool donors affects nodose ganglion (NG) neurophysiology or ii) gut microbial dysbiosis during inflammatory bowel disease (IBD) impacts the function of vagal afferent neurons.HypothesisRemodeling of gut microbiota during IBD increases secretion of mediators that change the excitability of vagal afferent neurons.MethodsTo examine the effect of IBD patients’ fecal supernatant (FS; 1:20 dilution) on the excitability of mouse vagal afferent neurons, NG neurons from C57/Bl6 mice were collected, dissociated, and incubated overnight with FS from 5 active Crohn’s disease (CD) patients, 7 active ulcerative colitis (UC) patients, and 5 healthy volunteers (HV). Current and voltage‐clamp recordings were used to assess changes in neuronal excitability and ion channel function.ResultsCD and UC FS significantly increased the excitability of NG neurons through a reduction in the rheobase of 40% (CD:60 cells vs. control: 54 cells), P<0.0001, Mann‐Whitney test) and 23% (n= 50 cells vs. control: 46 cells, P= 0.0038, Mann‐Whitney test) compared to their individual vehicle control neurons, respectively. This decrease in rheobase was accompanied by a two‐fold increase in the number of action potentials elicited at twice rheobase (P <0.01, Mann‐Whitney test). However, neither resting membrane potential, nor input resistance was altered in NG neurons treated with IBD FS compared with vehicle control neurons. HV FS had no effect on NG excitability. CD and UC FS significantly reduced voltage‐gated K+ currents (P= 0.0075 and P <0.0001, two‐way ANOVA followed by Sidak's multiple comparison test, respectively), but had no effect on voltage‐gated Na+ currents. The excitatory effect of CD and UC FS of NG neurons was blocked by the cysteine protease inhibitor (E64) (30 nM), but not the serine protease inhibitor (FUT175) (10 μM). In all CD patients, pre‐incubation of E64 blocked the increase in excitability by CD patient FS (CD rheobase:40.6 ± 3.6 pA vs.CD+ E64 rheobase: 73.9 ± 4.5 pA) (P<0.0001, one‐way ANOVA followed by Tukey's multiple comparison). However, E64 was only able to block the excitatory effect of FS from 5 out of 7 UC patients FS (UC rheobase:43.8 ± 4.7 pA vs. UC+ E64 rheobase: 83.5 ± 4.3 pA) (P=0.0092, one‐way ANOVA followed by Tukey's multiple comparison). The protease‐activated receptor 2 (PAR2) antagonist GB83 (10 μM) also blocked the effect of the CD and UC patient supernatant on NG neurons (P=0.0081and P=0.0116, Kruskal‐Wallis test, respectively).ConclusionFS from active IBD patients contain mediators that can excite NG neurons. Cysteine proteases directly mediates the effect of IBD FS on NG neurons by activation of PAR‐2. Signaling pathways downstream of PAR‐2 activation lead to inhibition of voltage‐gated K+ currents.

Rapid Intravenous Injection of Fentanyl (FNT) Causes a Vagal‐Mediated Lethal Sudden Ventilatory Arrest in Anesthetized Rats

Opioids have been used for analgesia and anesthesia mainly via activation of central opioid mu‐receptors (MORs). It also causes overdose deaths due to ventilatory failure, in which FNT is the deadest. Rapid intravenous (IV) injection of overdose FNT triggers a sudden death within a few minutes or even seconds, but the relevant mechanism remains unclear. Because bolus IV injection of FNT at a low dose produced a vagal‐mediated brief apnea (Zhuang et al, AJP 2012), This study was aimed to characterize the cardiorespiratory disorders responsible for the sudden death and determine the vagal role in generating the lethal ventilatory arrest. EMGs of external and internal intercostal (EMGEI and EMGII), thyroarytenoid and superior pharyngeal constrictor muscles (EMGTA and EMGSPC) were simultaneously recorded before and after IV injection of FNT in the anesthetized vagal intact and vagotomized rats. The optical fiber scope of an oral video camera was applied to visualize the vocal and pharyngeal responses. Immunohistochemical approach was used to define the presence of MOR expression in the pulmonary vagal sensory neurons retrogradely labeled by DiI. FNT induced an apnea or a lethal ventilatory arrest in a dose‐dependent manner associated with hypotension and bradycardia. The apnea/ventilatory arrest was characterized by silence of EMGEI with tonic discharges of EMGII, EMGTA, and EMGSPC, i.e., the central apnea (expiratory) coupled with vocal closure and pharyngeal constriction/collapse and chest wall rigidity (triple‐apnea). Vagotomy abolished the evoked cardiorespiratory responses. MOR expression in vagal pulmonary neurons, especially C‐neurons of the nodose and jugular ganglia. Our results suggest that rapid IV injection of FNT at a high dose uniquely causes a vagal‐mediated triple‐apnea, leading to the sudden death in anesthetized rats.

Optogenetic stimulation of cholinergic neuronal endings in the celiac‐superior mesenteric ganglion complex suppresses inflammation in murine endotoxemia

The vagus nerve plays a key role in the regulation of inflammation within the inflammatory reflex (Annu Rev Immunol, 2018, 36:783). In this major physiological neuroimmunoregulatory mechanism, efferent vagus nerve cholinergic axons interact with catecholaminergic neurons of the splenic nerve in the celiac‐superior mesenteric ganglion (CSMG) complex(Proc Natl Acad Sci USA, 2020, 117(47):29803).Previous studies utilizing electrical and optogenetic neuromodulation have demonstrated that vagus nerve cholinergic signalling suppresses pro‐inflammatory cytokine levels during endotoxemia and other inflammatory conditions. However, the specific role of cholinergic axonal terminals in the CSMG complex in this regulation remained enigmatic. To provide insight, we investigated the effect of selective optogenetic stimulation of cholinergic neuronal endings in the CSMG complex on cytokine and chemokine levels during murine endotoxemia. We used transgenic female 10–14‐week‐old mice expressing channelrhodopsin 2 coupled with yellow fluorescent protein on cholinergic, i.e., choline acetyltransferase positive neurons (ChAT‐ChR2‐eYFP). Anesthetized mice were subjected to abdominal surgery and the CSMG complex was localized medial to the upper pole left kidney and along the branch of the abdominal aorta, above the left renal vein. Blue laser light stimulation at 473nm was delivered in square pulses for 10mins at 20Hz, 25% duty cycle through an optic fiber cannula placed in contact with the CSMG complex. 30mins following optogenetic stimulation or sham stimulation, lipopolysaccharide (0.25mg/kg) was injected intraperitoneally and 90mins later mice were euthanized and blood, and spleen were collected and processed for cytokine and chemokines. Optogenetic stimulation of the CSMG complex in ChAT‐ChR2 mice (n=14) suppressed serum pro‐inflammatory cytokines and chemokines compared with sham stimulation (n=13), including TNF(401.7 ± 102.9 pg/ml vs 766.1 ± 207.2 pg/ml; P<0.0001) and MIP‐1α (6,523 ± 3,041 pg/ml vs 10,672 ± 3,915 pg/ml, P=0.0066), while the serum levels of the anti‐inflammatory cytokine IL‐10 were significantly increased (30,779 ± 10,035 pg/ml vs 22,979 ± 9,669 pg/ml, P=0.0427). There was also a significant reduction in splenic TNF levels in the stimulated vs the sham group(P=0.0023). Of note, optogenetic stimulation of the CSMG complex in littermate mice that do not express ChR2 on cholinergic neurons did not alter serum and splenic cytokine levels. These results provide the first experimental evidence that selective stimulation of cholinergic neuronal endings in the CSMG complex attenuates cytokine and chemokine levels. Future studies examining the specific contribution of vagal and sympathetic preganglionic cholinergic neurons in the CSMG complex in the regulation of immune responses will be instrumental for developing new targeted therapeutic anti‐inflammatory approaches.

Neurokinin 1 and 2 Receptors Differently Modulate PEG2‐ and Citric Acid‐Induced Cough and Ventilatory Responses

Inhalation of aerosolized citric acid (CA) or prostaglandin E2 (PGE2) provokes distinct cough patterns (Type I vs. II) and ventilatory responses (substantial hyperventilation vs. slight tachypnea) in humans and guinea pigs. Substance P (SP) and neurokinin A (NKA) are peripherally released mainly by vagal sensory fibers and capable of affecting CA‐induced cough and ventilatory response via preferentially activating neurokinin 1 and 2 receptors (NK1R and NK2R) respectively. The goal of this study was to determine the impacts of CA and PGE2 exposure on pulmonary SP and NKA levels and the roles of NK1R and NK2R in CA‐ and PGE2‐evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents after CA or PGE2 exposure for 10 min; (2) effects of vehicle, CP‐99994 and SR‐48968 (a NK1R and a NK2R antagonist) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on CA‐ and PGE2‐evoked cough and ventilation; and (3) immunoreactive expression of NK1R/NK2R in vagal sensory neurons labeled by TRPV1 (responsible for Type I cough) or EP3 receptors (responsible for Type II cough). CA and PGE2 exposure evoked Type I and II cough respectively associated with different degree of increases in pulmonary SP and NKA. CP‐99994 and SR‐48968 via both IP and IH similarly depressed the cough responses to CA with less impact on the cough response to PGE2 exposure and substantially inhibited or blocked the evoked ventilatory responses to both CA and PGE2. Moreover, NK1R and NK2R co‐expressed in vagal C‐neurons labeled by TRPV1 or EP3 receptors. These results suggest that the endogenously released SP and NKA play an important role in generating the cough and ventilatory responses to CA and maintaining the ventilatory response to PGE2, at least in part, via activation of vagal C‐neurons expressing NK1R and NK2R.