Spinal cord injury-mediated changes in electrophysiological properties of rat gastric nodose ganglion neurons.

In preclinical rodent models, spinal cord injury (SCI) manifests as gastric vagal afferent dysfunction both acutely and chronically. However, the mechanism that underlies this dysfunction remains unknown. In the current study, we examined the effect of SCI on gastric nodose ganglia (NG) neuron excitability and on voltage-gated Na+ (NaV) channels expression and function in rats after an acute (i.e. 3-days) and chronic (i.e. 3-weeks) period. Rats randomly received either T3-SCI or sham control surgery 3-days or 3-weeks prior to experimentation as well as injections of 3% DiI solution into the stomach to identify gastric NG neurons. Single cell qRT-PCR was performed on acutely dissociated DiI-labeled NG neurons to measure NaV1.7, NaV1.8 and NaV1.9 expression levels. The results indicate that all 3 channel subtypes decreased. Current- and voltage-clamp whole-cell patch-clamp recordings were performed on acutely dissociated DiI-labeled NG neurons to measure active and passive properties of C- and A-fibers as well as the biophysical characteristics of NaV1.8 channels in gastric NG neurons. Acute and chronic SCI did not demonstrate deleterious effects on either passive properties of dissociated gastric NG neurons or biophysical properties of NaV1.8. These findings suggest that although NaV gene expression levels change following SCI, NaV1.8 function is not altered. The disruption throughout the entirety of the vagal afferent neuron has yet to be investigated.

DT-0111: a novel drug-candidate for the treatment of COPD and chronic cough.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) plays important mechanistic roles in pulmonary disorders in general and chronic obstructive pulmonary disease (COPD) and cough in particular. The effects of ATP in the lungs are mediated to a large extent by P2X2/3 receptors (P2X2/3R) localized on vagal sensory nerve terminals (both C and Aδ fibers). The activation of these receptors by ATP triggers a pulmonary-pulmonary central reflex, which results in bronchoconstriction and cough, and is also proinflammatory due to the release of neuropeptides from these nerve terminals via the axon reflex. These actions of ATP in the lungs constitute a strong rationale for the development of a new class of drugs targeting P2X2/3R. DT-0111 is a novel, small, water-soluble molecule that acts as an antagonist at P2X2/3R sites. METHODS: Experiments using receptor-binding functional assays, rat nodose ganglionic cells, perfused innervated guinea pig lung preparation ex vivo, and anesthetized and conscious guinea pigs in vivo were performed. RESULTS: DT-0111 acted as a selective and effective antagonist at P2X2/3R, that is, it did not activate or block P2YR; markedly inhibited the activation by ATP of nodose pulmonary vagal afferents in vitro; and, given as an aerosol, inhibited aerosolized ATP-induced bronchoconstriction and cough in vivo. CONCLUSIONS: These results indicate that DT-0111 is an attractive drug-candidate for the treatment of COPD and chronic cough, both of which still constitute major unmet clinical needs. The reviews of this paper are available via the supplementary material section.

Opposing effects of bronchopulmonary C-fiber subtypes on cough in guinea pigs.

We have addressed the hypothesis that the opposing effects of bronchopulmonary C-fiber activation on cough are attributable to the activation of C-fiber subtypes. Coughing was evoked in anesthetized guinea pigs by citric acid (0.001-2 M) applied topically in 100-µl aliquots to the tracheal mucosa. In control preparations, citric acid evoked 10 ± 1 coughs cumulatively. Selective activation of the pulmonary C fibers arising from the nodose ganglia with either aerosols or continuous intravenous infusion of adenosine or the 5-HT3 receptor-selective agonist 2-methyl-5-HT nearly abolished coughing evoked subsequently by topical citric acid challenge. Delivering adenosine or 2-methyl-5-HT directly to the tracheal mucosa (where few if any nodose C fibers terminate) was without effect on citric acid-evoked cough. These actions of pulmonary administration of adenosine and 2-methyl-5-HT were accompanied by an increase in respiratory rate, but it is unlikely that the change in respiratory pattern caused the decrease in coughing, as the rapidly adapting receptor stimulant histamine also produced a marked tachypnea but was without effect on cough. In awake guinea pigs, adenosine failed to evoke coughing but reduced coughing induced by the nonselective C-fiber stimulant capsaicin. We conclude that bronchopulmonary C-fiber subtypes in guinea pigs have opposing effects on cough, with airway C fibers arising from the jugular ganglia initiating and/or sensitizing the cough reflex and the intrapulmonary C fibers arising from the nodose ganglia actively inhibiting cough upon activation.

Diet-induced obesity causes peripheral and central ghrelin resistance by promoting inflammation.

Ghrelin, a stomach-derived orexigenic peptide, transmits starvation signals to the hypothalamus via the vagus afferent nerve. Peripheral administration of ghrelin does not induce food intake in high fat diet (HFD)-induced obese mice. We investigated whether this ghrelin resistance was caused by dysfunction of the vagus afferent pathway. Administration (s.c.) of ghrelin did not induce food intake, suppression of oxygen consumption, electrical activity of the vagal afferent nerve, phosphorylation of ERK2 and AMP-activated protein kinase alpha in the nodose ganglion, or Fos expression in hypothalamic arcuate nucleus of mice fed a HFD for 12 weeks. Administration of anti-ghrelin IgG did not induce suppression of food intake in HFD-fed mice. Expression levels of ghrelin receptor mRNA in the nodose ganglion and hypothalamus of HFD-fed mice were reduced. Inflammatory responses, including upregulation of macrophage/microglia markers and inflammatory cytokines, occurred in the nodose ganglion and hypothalamus of HFD-fed mice. A HFD blunted ghrelin signaling in the nodose ganglion via a mechanism involving in situ activation of inflammation. These results indicate that ghrelin resistance in the obese state may be caused by dysregulation of ghrelin signaling via the vagal afferent.

Chronic exposure to low dose bacterial lipopolysaccharide inhibits leptin signaling in vagal afferent neurons.

Bacterially derived factors are implicated in the causation and persistence of obesity. Ingestion of a high fat diet in rodents and obesity in human subjects is associated with chronic elevation of low plasma levels of lipopolysaccharide (LPS), a breakdown product of Gram-negative bacteria. The terminals of vagal afferent neurons are positioned within the gut mucosa to convey information from the gut to the brain to regulate food intake and are responsive to LPS. We hypothesized that chronic elevation of LPS could alter vagal afferent signaling. We surgically implanted osmotic mini-pumps that delivered a constant, low-dose of LPS into the intraperitoneal cavity of rats (12.5 µg/kg/hr for 6 weeks). LPS-treated rats developed hyperphagia and showed marked changes in vagal afferent neuron function. Chronic LPS treatment reduced vagal afferent leptin signaling, characterized by a decrease in leptin-induced STAT3 phosphorylation. In addition, LPS treatment decreased cholecystokinin-induced satiety. There was no alteration in leptin signaling in the hypothalamus. These findings offer a mechanism by which a change in gut microflora can promote hyperphagia, possibly leading to obesity.

Blunted sympathoinhibitory responses in obesity-related hypertension are due to aberrant central but not peripheral signalling mechanisms.

The gut hormone cholecystokinin (CCK) acts at subdiaphragmatic vagal afferents to induce renal and splanchnic sympathoinhibition and vasodilatation, via reflex inhibition of a subclass of cardiovascular-controlling neurons in the rostroventrolateral medulla (RVLM). These sympathoinhibitory and vasodilator responses are blunted in obese, hypertensive rats and our aim in the present study was to determine whether this is attributable to (i) altered sensitivity of presympathetic vasomotor RVLM neurons, and (ii) aberrant peripheral or central signalling mechanisms. Using a diet-induced obesity model, male Sprague-Dawley rats exhibited either an obesity-prone (OP) or obesity-resistant (OR) phenotype when placed on a medium high fat diet for 13-15 weeks; control animals were placed on a low fat diet. OP animals had elevated resting arterial pressure compared to OR/control animals (P < 0.05). Barosensitivity of RVLM neurons was significantly attenuated in OP animals (P < 0.05), suggesting altered baroreflex gain. CCK induced inhibitory responses in RVLM neurons of OR/control animals but not OP animals. Subdiaphragmatic vagal nerve responsiveness to CCK and CCK1 receptor mRNA expression in nodose ganglia did not differ between the groups, but CCK induced significantly less Fos-like immunoreactivity in both the nucleus of the solitary tract and the caudal ventrolateral medulla of OP animals compared to controls (P < 0.05). These results suggest that blunted sympathoinhibitory and vasodilator responses in obesity-related hypertension are due to alterations in RVLM neuronal responses, resulting from aberrant central but not peripheral signalling mechanisms. In obesity, blunted sympathoinhibitory mechanisms may lead to increased regional vascular resistance and contribute to the development of hypertension.

Vagal afferents mediate antinociception of estrogen in a rat model of visceral pain: the involvement of intestinal mucosal mast cells and 5-hydroxytryptamine 3 signaling.

UNLABELLED: Estrogen reportedly facilitates visceral nociception at the spinal or supraspinal level. The present study was aimed to investigate whether estrogen modulates visceral pain through the vagal pathway. Ovariectomized rats received estradiol, which was administered subcutaneously (to act through both the vagal and spinal pathways) or intraduodenally (to preferentially act through the vagal pathway). Luminally applied estradiol induced a rapid and significant decrease in the visceromotor response to colorectal distension, with increased c-Fos expression in nodose ganglion neurons. Systemically injected estradiol increased visceromotor response and c-Fos expression in both nodose and dorsal root ganglion (T6-12) neurons. The antinociceptive effect of estrogen was abolished by surgical vagotomy or chemical denervation of vagal afferents. Both luminally and systemically administered estradiol elicited selective 5-hydroxytryptamine secretion from the duodenum. Granisetron, a 5-hydroxytryptamine 3 receptor antagonist, reversed the antinociceptive effect of estrogen. Intestinal mucosal mast cell stabilizers prevented estradiol-induced antinociception and 5-hydroxytryptamine secretion. Ultrastructural analysis revealed that estradiol caused piecemeal degranulation of intestinal mucosal mast cells. The actions of estradiol were inhibited by an estrogen receptor ß antagonist and mimicked by an estrogen receptor ß agonist. These results suggest that estrogen can trigger vagus-mediated antinociception, which is masked by its spinally mediated pronociception. PERSPECTIVE: This study is the first to show a vagus-mediated estrogenic antinociception, in which the nongenomic estrogen receptor ß-mediated, intestinal mucosal mast cell-derived 5-hydroxytryptamine/5-hydroxytryptamine 3 receptor pathway is involved. This work may provide new insights into the sex hormone modulation of visceral sensitivity related to irritable bowel syndrome and indicate potential therapeutic targets to manage this disease.

Vagal afferent innervation and remodeling in the aortic arch of young-adult fischer 344 rats following chronic intermittent hypoxia.

Previously, we have shown that chronic intermittent hypoxia (CIH) impairs baroreflex control of heart rate and augments aortic baroreceptor afferent function. In the present study, we examined whether CIH induces structural changes of aortic afferent axons and terminals. Young-adult Fischer 344 (F344, 4 months old) rats were exposed to room air (RA) or CIH for 35-45 days. After 14-24 days of exposure, they received tracer DiI injection into the left nodose ganglion to anterogradely label vagal afferent nerves. After surgery, animals were returned to their cages to continue RA or CIH exposure. Twenty-one days after DiI injection, the animals were sacrificed and the aortic arch was examined using confocal microscopy. In both RA and CIH rats, we found that DiI-labeled vagal afferent axons entered the wall of the aortic arch, then fanned out and branched into large receptive fields with numerous terminals (flower-sprays, end-nets and free endings). Vagal afferent axons projected much more to the anterior wall than to the posterior wall. In general, the flower-sprays, end-nets and free endings were widely and similarly distributed in the aortic arch of both groups. However, several salient differences between RA and CIH rats were found. Compared to RA control, CIH rats appeared to have larger vagal afferent receptive fields. The CIH rats had many abnormal flower-sprays, end-nets, and free endings which were intermingled and diffused into "bush-like" structures. However, the total number of flower-sprays was comparable (P>0.05). Since there was a large variance of the size of flower-sprays, we only sampled the 10 largest flower-sprays from each animal. CIH substantially increased the size of large flower-sprays (P<0.01). Numerous free endings with enlarged varicosities were identified, resembling axonal sprouting structures. Taken together, our data indicate that CIH induces significant remodeling of afferent terminal structures in the aortic arch of F344 rats. We suggest that such an enlargement of vagal afferent terminals may contribute to altered aortic baroreceptor function following CIH.

Blunted excitability of aortic baroreceptor neurons in diabetic rats: involvement of hyperpolarization-activated channel.

AIMS: Although dysfunction of arterial baroreflex occurs in human and animal models of type-1 diabetes (T1D), the mechanisms involved in the impairment of the baroreflex still remain unclear. The nodose ganglion (NG) contains the cell bodies of the aortic baroreceptor (AB) neurons. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in AB neurons and play an important role in regulating the cell excitability. We investigated whether the excitability of AB neurons is depressed in streptozotocin (STZ)-induced T1D rats and whether HCN channels are involved in this depression. METHODS AND RESULTS: Using the whole-cell patch clamp technique, we found that AB neuron excitability (action potential frequency at 50 pA current stimulation) in the T1D rats was lower than that in the sham rats (0.4 +/- 0.5 vs. 4.8 +/- 0.6 spikes/s, P < 0.05; AB neurons were identified by DiI staining). In addition, HCN current density in AB neurons from the T1D rats was bigger than that from the sham rats (60.2 +/- 6.1 vs. 30.7 +/- 4.9 pA/pF at test pulse -140 from holding potential -40 mV, P < 0.05). Furthermore, HCN channel blockers (5 mM cesium chloride and 100 microM ZD7288) significantly reduced HCN currents and increased action potential frequency of the AB neurons in sham and T1D rats. Immunofluorescent and western blot analyses demonstrated that the expression of HCN1 and HCN2 channel protein in the NG from the T1D rats was higher than that from the sham rats. CONCLUSION: These results indicate that the HCN channels influence the excitability of AB neurons, and more importantly, contribute to the decreased excitability of AB neurons in T1D rats.

Calcium transient evoked by nicotine in isolated rat vagal pulmonary sensory neurons.

It has been shown that inhaled cigarette smoke activates vagal pulmonary C fibers and rapidly adapting receptors (RARs) in the airways and that nicotine contained in the smoke is primarily responsible. This study was carried out to determine whether nicotine alone can activate pulmonary sensory neurons isolated from rat vagal ganglia; the response of these neurons was determined by fura-2-based ratiometric Ca(2+) imaging. The results showed: 1) Nicotine (10(-4) M, 20 s) evoked a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in 175 of the 522 neurons tested (Delta[Ca(2+)](i) = 142.2 +/- 12.3 nM); the response was reproducible, with a small reduction in peak amplitude in the same neurons when the challenge was repeated 20 min later. 2) A majority (59.7%) of these nicotine-sensitive neurons were also activated by capsaicin (10(-7) M). 3) 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP; 10(-4) M, 20 s), a selective agonist of the neuronal nicotinic acetylcholine receptors (NnAChRs), evoked a pattern of response similar to that of nicotine. 4) The responses to nicotine and DMPP were either totally abrogated or markedly attenuated by hexamethonium (10(-4) M). 5) In anesthetized rats, right atrial bolus injection of nicotine (75-200 mug/kg) evoked an immediate (latency <1-2 s) and intense burst of discharge in 47.8% of the pulmonary C-fiber endings and 28.6% of the RARs tested. In conclusion, nicotine exerts a direct stimulatory effect on vagal pulmonary sensory nerves, and the effect is probably mediated through an activation of the NnAChRs expressed on the membrane of these neurons.

Endotracheal tube electrodes to map and monitor activities of the vagus nerve intraoperatively. Technical note.

Difficulty swallowing due to damage of the vagus nerve is one of the most devastating complications of surgery in and around the medulla oblongata; therefore, intraoperative anatomical and functional evaluation of this nerve is crucial. The authors applied endotracheal tube surface electrodes to record electromyography (EMG) activity from vocal cords innervated by the vagus nerve. The vagal nucleus or rootlet was electrically stimulated during surgery and vocalis muscle EMG activities were displayed by auditory and visual signals. This technique was used successfully to identify the vagus motor nerve and evaluate its integrity during surgery. The advantages of this method compared with the use of needle electrodes include safe simple electrode placement and stable recording during surgery. In cases involving a pontine cavernoma pressing the nucleus or a jugular foramen tumor encircling the rootlet, this method would be particularly valuable. Additional studies with a larger number of patients are needed to estimate the significance of this method as a means of functional monitoring to predict clinical function.

Autotransplantation of peripheral cholinergic neurons into the brains of Alzheimer model rats.

Current hypotheses regarding Alzheimer's disease implicate cholinergic function. In this study, peripheral cholinergic neurons in the vagal nodosal ganglion were transplanted into the brains of Alzheimer model rats. Eighteen Sprague-Dawley strain rats were divided into three groups: 1) unoperated control rats, 2) rats that had undergone bilateral destruction of the nucleus basalis of Meynert (NBM) (Alzheimer model), and 3) the transplantation group in which the vagal nodosal ganglion was transplanted into the cerebral neocortex one week after the bilateral destruction of the Meynert nucleus. Seven weeks after the transplantation rat behaviour was assessed using psychological tests (spontaneous activity, passive avoidance response and the Hebb-Williams maze test). The Alzheimer model rats had a statistically significant increase in spontaneous activity in comparison with controls (P less than 0.01). The transplant rats showed some amelioration of this abnormal increase in spontaneous activity observed in the Alzheimer model rats. All of the control rats showed conditioned passive avoidance responses, while only one Alzheimer model rat retained is shocked-conditions behaviour before 24 hours (P less than 0.01). Three of the six transplanted rats showed complete improvement in the passive avoidance response test. In the Hebb-Williams maze test, the rats with NMB lesions made more errors than the control rats. The transplanted rats had a lower number of errors than NBM-lesioned rats but still more than the controls. Histological examination revealed many cholinergic cells in the transplanted tissue, especially in the area adjacent to the cerebral cortical surface. The present results indicate that autotransplantation of peripheral cholinergic cells ameliorates abnormal behaviour in Alzheimer model rats.