Non-invasive stimulation of vagal afferents reduces gastric frequency

Metabolic feedback between the gut and the brain relayed via the vagus nerve contributes to energy homeostasis. We investigated in healthy adults whether non-invasive stimulation of vagal afferents impacts energy homeostasis via efferent effects on metabolism or digestion. In a randomized crossover design, we applied transcutaneous auricular vagus nerve stimulation (taVNS) while recording efferent metabolic effects using simultaneous electrogastrography (EGG) and indirect calorimetry. We found that taVNS reduced gastric myoelectric frequency (p = .008), but did not alter resting energy expenditure. We conclude that stimulating vagal afferents induces gastric slowing via vagal efferents without acutely affecting net energy expenditure at rest. Collectively, this highlights the potential of taVNS to modulate digestion by activating the dorsal vagal complex. Thus, taVNS-induced changes in gastric frequency are an important peripheral marker of brain stimulation effects.     

Antidromic activation of vagal and sympathetic afferents does not produce intestinal contractions in dogs.

The question has been asked whether vagal and sympathetic afferents activated antidromically play a role as motor nerves on the in vivo small intestine in dogs anesthetized with urethane. The vagus nerve of one side was cut above the nodose ganglion and the efferent fibers allowed to degenerate. Peripheral stimulation (5-50 Hz, 0.5-3 ms, 5-25 V) of an intact cervical vagus, being able to excite both efferent and afferent fibers, caused large contractions in the jejunum and stomach, whereas stimulation of the contralateral cut cervical vagus could not produce any response in the jejunum but small contractions in the stomach. Peripheral stimulation of the cut cervical vagus did not produce bradycardia and hypotension. Single- and multi-unit discharges to distension of the jejunal segments could be recorded from the peripheral cut end of the cut cervical vagus. Immunohistochemically, there were many substance P-containing cells in both nodose ganglia. Antidromic stimulation of the dorsal roots (T7-T10) did not induce any response in the jejunum but contractions in the stomach. The results may confirm that vagal and sympathetic afferents have no antidromic motor function at least in the in vivo canine small intestine.     

Receptor-mediated activation of gastric vagal afferents by glucagon-like peptide-1 in the rat

Abstract  The vagus nerve plays a role in mediating effects of the two glucagon-like peptides GLP-1 and GLP-2 on gastrointestinal growth, functions and eating behaviour. To obtain electrophysiological and molecular evidence for the contribution of afferent pathways in chemoreception from the gastrointestinal tract, afferent mass activity in the ventral gastric branch of the vagus nerve and gene expression of GLP-1 receptors and GLP-2 receptors in the nodose ganglion were examined in Sprague–Dawley rats. Intravenous administration of GLP-1 (30–1000 pmol kg⁻¹), reaching high physiological plasma concentrations, increased vagal afferent mass activity peaking (13–52% above basal level, P  <   0.05) 3–5 min after injection. Repeated administration of GLP-1 (1000 pmol kg⁻¹; five times, 15 min intervals) elicited similar responses. Pretreatment with GLP-1 receptor antagonist exendin(9-39)amide (500 pmol kg⁻¹) abolished the GLP-1 response to doses 30–300 pmol kg⁻¹ but had no effect on the vagal response to gastric distension. For comparison, GLP-2 (1000 pmol kg⁻¹) had no effect on vagal afferent activity. Vagal chemoreception of GLP-1 is supported by expression of the GLP-1 receptor gene in the nodose ganglion. However, the GLP-2 receptor was also expressed. To conclude, our results show that peripherally administered GLP-1, differently from GLP-2, activates vagal afferents, with no evidence of desensitisation. The GLP-1 effect was blocked by exendin(9-39)amide, suggesting that GLP-1 receptors on vagal afferent nerves mediate sensory input from the gastrointestinal tract or pancreas; either directly or indirectly via the release of another mediator. GLP-2 receptors appear not be functionally expressed on vagal afferents.     

Excitation of dorsal motor vagal neurons evokes non-nicotinic receptor-mediated gastric relaxation

Vagal stimulation results in both gastric motor excitatory and non-adrenergic non-cholinergic (NANC) inhibitory responses. The NANC pathway involves preganglionic cholinergic neurons, which act through nicotinic receptors to ultimately evoke gastric smooth muscle relaxation via release of nitric oxide (NO) and other neurotransmitters. Within the dorsal motor nucleus of the vagus (DMN), some preganglionic neurons also contain NO synthase. The NO synthase-containing neurons innervate the gastric fundus where adaptive relaxation occurs. This study tests the hypothesis that chemical stimulation of vagal motor neurons in animals, in which nicotinic receptors are blocked, evokes an NO-dependent gastric relaxation. A cell body excitant, N-methyl-D-aspartate (NMDA, 0.03-3 nmol), was microinjected into the DMN in anesthetized rats while recording intragastric pressure (IgP). The first group received NMDA before and after administration of a ganglionic blocker, hexamethonium bromide (15 mg/kg, i.v.) and atropine (1.0 mg/kg). Significant dose-dependent increases in IgP and gastric motility occurred before hexamethonium after the 0.3 and 3 nmol doses of NMDA. After hexamethonium, 0.3 and 3 nmol NMDA evoked significant decreases in IgP. A second group of rats was hexamethonium-pretreated and received NMDA microinjection into the DMN before and after an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (10 mg/kg, i.v.). The NMDA-evoked decrease in IgP was completely abolished by the NO synthase inhibitor. These data support the novel idea that NO synthase-containing preganglionic neurons mediate gastric relaxation that is independent of nicotinic receptors.     

Selective retrograde transport of H-serotonin in vagal afferents

A new serotonergic afferent vagal component has been demonstrated in the cat by radioautography. Twenty-four hours after a bilateral injection of tritiated serotonin (³H-5-HT) into the area of the nucleus of the solitary tract (NST), heavily and lightly labelled cell bodies were observed in the nodose ganglia. After unilateral injections of ³H-5-HT into the same area, labelled ganglionar cell bodies were found in the ipsilateral nodose ganglion. Some were also found in the contralateral one, suggesting a serotonergic crossed fibers component. Dense clusters of silver grains, depicting typical labelling of neuronal varicosities, were observed in the NST. After destruction of the serotonergic terminals with 5,7-dihydroxytryptamine, followed by injection of ³H-5-HT, the number of labelled cell bodies decreased dramatically in the ipsilateral nodose ganglia and the clusters of silver grains disappeared in the NST. After ligature or section of the supranodose vagal nerve, following injection of ³H-5-HT into the NST, no radioautographic reaction was observed in the homolateral nodose ganglia. The present study demonstrates the existence of a peripheral serotonergic system in vagal afferents. The physiological implications of this new serotonergic visceral pathway remain to be studied.     

Gastric electrical stimulation at proximal stomach induces gastric relaxation in dogs

Gastric electrical stimulation (GES) improves symptoms in patients with gastroparesis. However, the underlying mechanisms remain unclear. To determine if GES at proximal and distal stomach could affect the biomechanical properties of the stomach, thus contributing to the beneficial effect of GES. Four pairs of electrodes were implanted along the greater curvature of the stomach in seven dogs. Gastric tone and compliance was assessed with a barostat. Measurements were obtained randomly during control and proximal and distal stimulation (4 mA, 375 ms and 6/18 cpm). Data as mean or median (25-75th percentiles). Gastric compliance was not affected by proximal and distal GES. Gastric tone was significantly reduced during proximal GES: 82.0 (66.8, 89.1) mL vs control 49.7 (39.6,75.9) mL at 6 cpm (P = 0.016), and 90.6 (54.5, 117.9) mL vs control 62.8 (39.6, 75.9) mL at 18 cpm (P = 0.031). Tone was not affected by distal GES at 6 cpm: 95.8 (46.3, 106.7) mL vs control 75.2 (49.7, 86.1) mL (P = 0.47) and at 18 cpm: 80.4 (38.1, 170.3) mL vs control 62.8 (44.6, 156.3) mL (P = 0.44). Proximal GES induces gastric relaxation. This effect, if seen also in humans, may explain, in part, the symptomatic improvement associated with GES therapy in patients with gastroparesis.     

Withdrawal and restoration of central vagal afferents within the dorsal vagal complex following subdiaphragmatic vagotomy

Vagotomy, a severing of the peripheral axons of the vagus nerve, has been extensively utilized to determine the role of vagal afferents in viscerosensory signaling. Vagotomy is also an unavoidable component of some bariatric surgeries. Although it is known that peripheral axons of the vagus nerve degenerate and then regenerate to a limited extent following vagotomy, very little is known about the response of central vagal afferents in the dorsal vagal complex to this type of damage. We tested the hypothesis that vagotomy results in the transient withdrawal of central vagal afferent terminals from their primary central target, the nucleus of the solitary tract (NTS). Sprague–Dawley rats underwent bilateral subdiaphragmatic vagotomy and were sacrificed 10, 30, or 60 days later. Plastic changes in vagal afferent fibers and synapses were investigated at the morphological and functional levels by using a combination of an anterograde tracer, synapse‐specific markers, and patch‐clamp electrophysiology in horizontal brain sections. Morphological data revealed that numbers of vagal afferent fibers and synapses in the NTS were significantly reduced 10 days following vagotomy and were restored to control levels by 30 days and 60 days, respectively. Electrophysiology revealed transient decreases in spontaneous glutamate release, glutamate release probability, and the number of primary afferent inputs. Our results demonstrate that subdiaphragmatic vagotomy triggers transient withdrawal and remodeling of central vagal afferent terminals in the NTS. The observed vagotomy‐induced plasticity within this key feeding center of the brain may be partially responsible for the response of bariatric patients following gastric bypass surgery. J. Comp. Neurol. 521:3584‐3599, 2013. © 2013 Wiley Periodicals, Inc.     

Atropine prevents the reflex tracheal relaxation arising from the stimulation of intestinal and skeletal muscle afferents in dogs

Using cholinergic and beta-adrenergic antagonists, we characterized the efferent arms of two reflex arcs that relax tracheal smooth muscle in dogs. The reflex arcs investigated were activated by the capsaicin-induced stimulation of afferent endings in the intestine and in the hindlimb. We found that the reflex tracheal relaxations were due to a withdrawal of a tonic cholinergic input. Beta-adrenergic pathways played little role in causing these reflex responses.     

Contribution of acetylcholine,vasoactive intestinal polypeptide and nitric oxide to CNS-evoked vagal gastric relaxation in the rat

Abstract Several in vitro models of gastric relaxation have elucidated a role of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in non-adrenergic, non-cholinergic (NANC) vagally mediated gastric relaxation. However, these models do not necessarily mimic the events leading to gastric relaxation in the whole animal. We have recently described a vagally mediated gastric relaxation evoked by micro-injection of substance P (SP) into the nucleus raphe obscurus (NRO). The present study was performed to elucidate whether this CNS-stimulated in vivo gastric relaxation involved acetylcholine, NO and VIP. Atropine (1 mg kg^?1 i.v.), reduces both the rapid nadir and sustained gastric relaxation evoked by SP in the NRO, and the residual responses are abolished by N^G-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 10 mg kg^?1 i.v.), an NO synthase inhibitor. Blockade of NO synthase alone is not sufficient to abolish the effect of SP into the NRO on intragastric pressure. A VIP antagonist, [p-chloro-D-Phe⁶, Leu¹⁷]VIP (32 μg i.v.) alone, or with the addition of L-NAME, does not affect the nadir of the gastric relaxation in response to SP microinjected into the NRO; however, both antagonists reduce the CNS-evoked sustained intragastric pressure relaxation. We conclude that, in CNS-evoked gastric relaxation, inhibition of cholinergic pathways is potentially important for both the rapid nadir and sustained gastric relaxation, and both NO and VIP contribute to sustained gastric relaxation.     

Genetic tracing of Nav1.8-expressing vagal afferents in the mouse

Nav1.8 is a tetrodotoxin-resistant sodium channel present in large subsets of peripheral sensory neurons, including both spinal and vagal afferents. In spinal afferents, Nav1.8 plays a key role in signaling different types of pain. Little is known, however, about the exact identity and role of Nav1.8-expressing vagal neurons. Here we generated mice with restricted expression of tdTomato fluorescent protein in all Nav1.8-expressing afferent neurons. As a result, intense fluorescence was visible in the cell bodies, central relays, and sensory endings of these neurons, revealing the full extent of their innervation sites in thoracic and abdominal viscera. For instance, vagal and spinal Nav1.8-expressing endings were seen clearly within the gastrointestinal mucosa and myenteric plexus, respectively. In the gastrointestinal muscle wall, labeled endings included a small subset of vagal tension receptors but not any stretch receptors. We also examined the detailed innervation of key metabolic tissues such as liver and pancreas and evaluated the anatomical relationship of Nav1.8-expressing vagal afferents with select enteroendocrine cells (i.e., ghrelin, glucagon, GLP-1). Specifically, our data revealed the presence of Nav1.8-expressing vagal afferents in several metabolic tissues and varying degrees of proximity between Nav1.8-expressing mucosal afferents and enteroendocrine cells, including apparent neuroendocrine apposition. In summary, this study demonstrates the power and versatility of the Cre-LoxP technology to trace identified visceral afferents, and our data suggest a previously unrecognized role for Nav1.8-expressing vagal neurons in gastrointestinal functions.     

Electrical and chemical stimulation of the nucleus raphe magnus inhibits induction of retching by afferent vagal fibers

Like pain sensation, vomiting is induced by C afferent fibers of the vagus nerve. Therefore, it can be assumed to be suppressed by the descending inhibitory system (DIS) of pain. In this study we examined this possibility in immobilized decerebrated dogs. Fictive retching was induced by the stimulation of abdominal afferent vagal fibers (stim-abdVAs) and identified by activity patterns of efferent discharges of the phrenic and abdominal muscle nerves. The induction of retching was suppressed by weak stimulation (20 μA, 20 Hz, 0.5 ms duration) of the pontine area corresponding to the nucleus raphe magnus (NRM). Stronger suppressions were produced by stimulation of the rostral part of the NRM at the pontine level where the abducen nucleus exists. Pontamine sky blue, an excitatory neurotoxin, which was microinjected into the NRM area in 5 dogs, completely suppressed the induction of retching during the injection period. The NRM area at the level of the abducen nucleus was commonly stained by the dye in the 5 dogs. These results suggest that the DIS of pain suppresses the induction of retching by afferent vagal fibers.     

Pulmonary vagal sensory afferents and spontaneous EEG rhythms in the cat sensorimotor cortex

Interactions between vagal afferent fibres and spontaneous electroencephalographic (EEG) activity, recorded on the sensorimotor cortex of the cat, were studied during the mechanical activation of pulmonary afferents. The interactions were compared to the cortical effects of the electrical stimulation of all vagal fibers or to the chemical activation of unmyelinated vagal afferents (C-fibers) by phenyldiguanide. The present study was performed on anesthetized cats, artificially ventilated with open chest. Over 60 locations were explored on the posterior sigmoid gyrus. Repetitive electrical stimulation (30 Hz, 0.8 ms shock duration) of the contralateral cervical vagus nerve or of both nerves induced within less than 5 s changes in the pattern and periodicity of EEG spindles, associated with depressed background rhythms or rhythmic EEG activities. Cortical responses were also observed after i.v. injection of phenyldiguanide. Changes in activity of pulmonary stretch receptors by lung hyperinflation or suppression of phasic lung inflations ('stop pump') had no effect on the EEG rhythms. On the other hand, expiratory threshold loading or passive hyperdeflation of the lungs elicited EEG changes similar to those obtained by electrical stimulation of all vagal fibers. After bilateral vagotomy, all these responses disappeared or were delayed. The present observations strongly suggest that sensory information carried by thin vagal fibers greatly influences cortical rhythms in the cat sensorimotor cortex.     

Activation of cardiac vagal afferents facilitates late vagal inhibition in neurones of the rostral ventrolateral medulla oblongata bilaterally

A reduced activity of cardiac vagal afferent fibres is considered as one of the pathophysiological causes of post-infarction complications [A. Head, Baroreflexes and cardiovascular regulation in hypertension. J. Cardiovasc. Pharmacol. 26 (1995) S7-S16]. The mechanism of how a reduction of cardiac vagal activity leads to enhanced sympathetic drive and systemic hypertension is however not yet clear. Experimental data have shown that the rostral ventrolateral medulla oblongata (RVLM) plays an important role in tonic blood pressure regulation, the control of sympathetic vasoconstriction and cardiac performance. The aim of the study was to determine whether activation of cardiac vagal afferents contributes to eliciting the long-lasting late inhibition that we have previously shown to occur in neurones of the RVLM [A. Zagon, K. Ishizuka, I. Rocha, K.M. Spyer, Late vagal inhibition in neurones of the ventrolateral medulla oblongata in the rat. Neurosci. 92 (1999) 877-888]. The experiments were carried out in terminally anaesthetised and artificially ventilated rats using in vivo intracellular recordings. The data confirmed that late vagal inhibition is elicited by cumulative activation of functionally different vagal afferents, including those that originate from cardiac receptors. It was also demonstrated that activation of cardiac afferents could lead to a significant increase in the duration of this long-lasting late response component. Facilitation of late vagal inhibition was observed in RVLM neurones both ipsi- and contralateral to the stimulated nerve. It is suggested that such facilitation of late vagal inhibition may be a mechanism of how pulse-synchronous activation of cardiac afferents leads to a tonic modulation of the activity of RVLM neurones. An attenuation of late vagal inhibition during reduced activity of cardiac vagal afferents could lead to enhanced excitability in these neurones which in turn can lead to an increase in medullary sympathetic outflows towards the heart and peripheral blood vessels.     

Gastric secretion but not nitric oxide is involved in pentagastrin-induced gastric relaxation in conscious dogs

Gastrin delays gastric emptying of liquids probably by reducing gastric tone. The mechanism responsible for the relaxatory effect induced by pentagastrin was unknown. The aim of this study was to investigate the effects of pentagastrin and the underlying mechanisms responsible for these effects. Could nitric oxide (NO) be involved as a mediator? Gastric tone was monitored with a flaccid bag introduced into the stomach via a gastric cannula and connected to a barostat. A series of pressure–volume curves with a 30-min interval were constructed by increasing intragastric pressure to a maximum of 14 mmHg (2-mmHg steps). Pentagastrin (4 μg kg^?1 s.c.) facilitated the volume increases induced by isobaric gastric distension. This effect could be completely blocked by pretreatment with cimetidine (10 mg kg^?1 s.c.) or by omeprazole (10 mg kg^?1 p.o.). The effect induced by pentagastrin could be mimicked by histamine (0.16 mg kg^?1 s.c.) and to a lesser extent by insulin (0.2 IU kg^?1 i.v.). Cimetidine and omeprazole had no intrinsic effect on gastric tone. With an opened cannula, allowing the gastric secretions to leave the stomach, no increased gastric relaxation could be observed either in the presence of pentagastrin or in the presence of histamine or insulin. Nitro-l-arginine (L-NNA, 5 mg kg^?1 i.v.) reduced the volume increases induced by distension. Unexpectedly, even in the presence of L-NNA, pentagastrin remained effective. In conclusion, pentagastrin induces a gastric relaxation via a mechanism which involves gastric secretion but not nitric oxide.     

Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius

Combined radioautography and immunocytochemistry were used to define the ultrastructure and synaptic relations between vagal sensory afferents and catecholaminergic (CA) neurons of the A2 group located within the nucleus tractus solitarius (NTS) of rat brain. The vagal afferents were radioautographically labeled by tritiated amino acids anterogradely transported from the nodose ganglion. Immunocytochemical labeling for tyrosine hydroxylase (TH) served for the identification of catecholaminergic neurons. The radiographically labeled axons seen by light microscopy were widely distributed throughout the more caudal NTS. The reduced silver grains were more densely distributed within the NTS located homolateral to the injected nodose ganglion. The radioautographically labeled processes were localized in regions containing catecholaminergic neurons as indicated by immunoreactivity for TH. Electron microscopic analysis of the medial NTS at the level of the obex demonstrated that the reduced silver grains were localized within axon terminals. The radioautographically labeled terminals were 2-3 microns in diameter, contained numerous small, clear and a few large, dense vesicles, and formed predominately axodendritic synapses. Many of the recipient dendrites contained immunoreactivity for TH. In rare instances, vagal afferents formed synaptic appositions with both TH-labeled and unlabeled axon terminals and neuronal soma. This study provides the first ultrastructural evidence that the catecholaminergic neurons within the NTS receive direct synapses from sensory neurons in the nodose ganglion.     

A novel preparation to study rat pancreatic spinal and vagal mechanosensitive afferents in vitro

Abstract  The management of pancreatic pain is a significant clinical problem so understanding of how sensory signals are generated in pancreatic tissue is fundamental. We aimed to characterize mechanosensitive and chemosensitive properties of pancreatic spinal and vagal afferents in vitro . Spinal and vagal afferent preparations from Sprague–Dawley rats were established incorporating the left splanchnic nerve or vagus nerves respectively. The common bile duct was cannulated for distension of the pancreatic duct with fluid. Nerve discharge evoked by blunt probing, duct distension or electrical stimulation was obtained from teased nerve bundles using standard extra-cellular recording. Discharge from 197 spinal afferent bundles was recorded, of which 57% displayed spontaneous activity. Blunt probing revealed 61 mechanosensitive receptive fields which were associated primarily with arteries/blood vessels (33/61) and the parenchyma (22/61). All mechanosensitive responses were slowly adapting, with 33% continuing to discharge after termination of the stimulus and 60% displaying a response threshold <10 g. Application of chemical mediators (bradykinin, histamine, 5-hydroxytryptamine, cholecystokinin octapeptide) evoked a response from 31/57 units, with 33% excitatory and 23% inhibitory. Spontaneous discharge was recorded from 72% of 135 vagal bundles. Mechanosensitive receptive fields were not identified in the pancreas but were evident in adjacent organs. No spinal or vagal afferent response to duct distension was obtained. In conclusion, pancreatic mechanosensitive spinal afferents are common, in contrast to pancreatic mechanosensitive vagal afferents indicating that pancreatic sensory innervation is predominantly spinal. Chemosensitive spinal afferent nerve endings are present in the pancreas and respond to a variety of inflammatory and physiological mediators.     

Cardiopulmonary sympathetic and vagal afferents excite C1-C2 propriospinal cells in rats

The purpose of this study in anesthetized rats was to determine the effects of stimulating cardiopulmonary sympathetic afferents (CPSA) and vagal afferents on C1-C2 descending propriospinal neurons. We hypothesized that inhibition of spinal sensory neurons produced by CPSA or vagus activation might relay in C1-C2 spinal segments. Extracellular action potentials were recorded from 73 C1-C2 neurons whose axons were antidromically activated in lumbar segments. CPSA input excited 22 cells, inhibited two cells and excited/inhibited one cell, whereas vagal input excited eight cells and inhibited two cells. Results are consistent with the hypothesis that CPSA input can be processed in C1-C2 segments to produce neural modulation in distant spinal segments.