Role of nerve growth factor in modulation of gastric afferent neurons in the rat

Recent studies demonstrated that experimental ulcers are associated with changes in the properties of voltage-sensitive sodium currents in sensory neurons. We hypothesized that nerve growth factor (NGF) contributes to these changes. Gastric ulcers were induced by acetic acid injection into the wall of the rat stomach. NGF expression was determined by ELISA and immunohistochemically. Sensory neurons were labeled by injection of a retrograde tracer into the gastric wall. Sodium currents were recorded in gastric sensory neurons from nodose and dorsal root ganglia cultured for 24 h in the presence of NGF or a neutralizing NGF antibody, respectively. Gastric ulcer formation caused a rise in NGF concentration within the gastric wall and an increase in NGF immunoreactivity. Exposure to NGF caused a significant increase in the TTX-resistant sodium current, whereas the TTX-sensitive sodium current remained unchanged. This was associated with an acceleration of the recovery from inactivation in spinal sensory neurons. Production and release of NGF in the gastric wall may contribute to sensitization of primary afferent neurons during gastric inflammation.     

Nesfatin-1 inhibits gastric acid secretion via a central vagal mechanism in rats

Nesfatin-1, a novel hypothalamic peptide, inhibits nocturnal feeding behavior and gastrointestinal motility in rodents. The effects of nesfatin-1 on gastrointestinal secretory function, including gastric acid production, have not been evaluated. Nesfatin-1 was injected into the fourth intracerebral ventricle (4V) of chronically cannulated rats to identify a nesfatin dose sufficient to inhibit food intake. Nesfatin-1 (2 μg) inhibited dark-phase food intake, in a dose-dependent fashion, for >3 h. Gastric acid production was evaluated in urethane-anesthetized rats. Nesfatin-1 (2 μg) was introduced via the 4V following endocrine stimulation of gastric acid secretion by pentagastrin (2 μg·kg(-1)·h(-1) iv), vagal stimulation with 2-deoxy-d-glucose (200 mg/kg sc), or no stimulus. Gastric secretions were collected via gastric cannula and neutralized by titration to determine acid content. Nesfatin-1 did not affect basal and pentagastrin-stimulated gastric acid secretion, whereas 2-deoxy-d-glucose-stimulated gastric acid production was inhibited by nesfatin-1 in a dose-dependent manner. c-Fos immunofluorescence in brain sections was used to evaluate in vivo neuronal activation by nesfatin-1 administered via the 4V. Nesfatin-1 caused activation of efferent vagal neurons, as evidenced by a 16-fold increase in the mean number of c-Fos-positive neurons in the dorsal motor nucleus of the vagus (DMNV) in nesfatin-1-treated animals vs. controls (P < 0.01). Finally, nesfatin-induced Ca(2+) signaling was evaluated in primary cultured DMNV neurons from neonatal rats. Nesfatin-1 caused dose-dependent Ca(2+) increments in 95% of cultured DMNV neurons. These studies demonstrate that central administration of nesfatin-1, at doses sufficient to inhibit food intake, results in inhibition of vagally stimulated secretion of gastric acid. Nesfatin-1 activates DMNV efferent vagal neurons in vivo and triggers Ca(2+) signaling in cultured DMNV neurons.     

Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways

In the vagal-sensory system, neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) are synthesized nearly exclusively in small-diameter nociceptive type C-fiber neurons. By definition, these neurons are designed to respond to noxious or tissue-damaging stimuli. A common feature of visceral inflammation is the elevation in production of sensory neuropeptides. Little is known, however, about the physiological characteristics of vagal sensory neurons induced by inflammation to produce substance P. In the present study, we show that allergic inflammation of guinea pig airways leads to the induction of substance P and CGRP production in large-diameter vagal sensory neurons. Electrophysiological and anatomical evidence reveals that the peripheral terminals of these neurons are low-threshold Adelta mechanosensors that are insensitive to nociceptive stimuli such as capsaicin and bradykinin. Thus inflammation causes a qualitative change in chemical coding of vagal primary afferent neurons. The results support the hypothesis that during an inflammatory reaction, sensory neuropeptide release from primary afferent nerve endings in the periphery and central nervous system does not require noxious or nociceptive stimuli but may also occur simply as a result of stimulation of low-threshold mechanosensors. This may contribute to the heightened reflex physiology and pain that often accompany inflammatory diseases.     

Capsaicin and the stomach. A review of experimental and clinical data

Capsaicin, the pungent principle of hot pepper, because of its ability to excite and later defunctionalize a subset of primary afferent neurons, has been extensively used as a probe to elucidate the function of these sensory neurons in a number of physiological processes. In the rat stomach, experimental data provided clear evidence that capsaicin-sensitive (CS) sensory nerves are involved in a local defense mechanism against gastric ulcer. Stimulation of CS sensory nerves with low intragastric concentrations of capsaicin protected the rat gastric mucosa against injury produced by different ulcerogenic agents. High local desensitizing concentrations of capsaicin or systemic neurotoxic doses of the agent markedly enhanced the susceptibility of the rat gastric mucosa to later noxious challenge. Resiniferatoxin, a potent analogue of capsaicin possesses an acute gastroprotective effect similar to that of capsaicin in the stomach. The gastroprotective effect of capsaicin-type agents involves an enhancement of the microcirculation effected through the release of mediator peptides from the sensory nerve terminals with calcitonin gene-related peptide being the most likely candidate implicated. They do not depend on vagal efferent or sympathetic neurons or involve prostanoids. The gastric mucosal protective effect of prostacyclin is retained after systemic or topical capsaicin desensitization. Capsaicin-sensitive fibers are involved in the repair mechanisms of the gastric mucosa. A protective role for CS sensory nerves has also been demonstrated in the colon. In most studies, capsaicin given into the stomach of rats or cats inhibited gastric acid secretion. In humans, although recent studies provide evidence in favor of a beneficial effect of capsaicin on the gastric mucosa, an exact concentration-related assessment of the effect of the agent is still lacking.     

Noradrenergic neurons in the rat solitary nucleus participate in the esophageal-gastric relaxation reflex

Activation of esophageal mechanosensors excites neurons in and near the central nucleus of the solitary tract (NSTc). In turn, NSTc neurons coordinate the relaxation of the stomach [i.e., the receptive relaxation reflex (RRR)] by modulating the output of vagal efferent neurons of the dorsal motor nucleus of the vagus (DMN). The NSTc area contains neurons with diverse neurochemical phenotypes, including a large population of catecholaminergic and nitrergic neurons. The aim of the present study was to determine whether either one of these prominent neuronal phenotypes was involved in the RRR. Immunohistochemical techniques revealed that repetitive esophageal distension caused 53% of tyrosine hydroxylase-immunoreactive (TH-ir) neurons to colocalize c-Fos in the NSTc. No nitric oxide synthase (NOS)-ir neurons in the NSTc colocalized c-Fos in either distension or control conditions. Local brain stem application (2 ng) of alpha-adrenoreceptor antagonists (i.e., alpha1-prazosin or alpha2-yohimbine) significantly reduced the magnitude of the esophageal distension-induced gastric relaxation to approximately 55% of control conditions. The combination of yohimbine and prazosin reduced the magnitude of the reflex to approximately 27% of control. In contrast, pretreatment with either the NOS-inhibitor NG-nitro-l-arginine methyl ester or the beta-adrenoceptor antagonist propranolol did not interfere with esophageal distension-induced gastric relaxation. Unilateral microinjections of the agonist norepinephrine (0.3 ng) directed at the DMN were sufficient to mimic the transient esophageal-gastric reflex. Our data suggest that noradrenergic, but not nitrergic, neurons of the NSTc play a prominent role in the modulation of the RRR through action on alpha1- and alpha2-adrenoreceptors. The finding that esophageal afferent stimulation alone is not sufficient to activate NOS-positive neurons in the NSTc suggests that these neurons may be strongly gated by other central nervous system inputs, perhaps related to the coordination of swallowing or emesis with respiration.     

Selective potentiating effect of RS14203 on a serotoninergic pathway in anesthetized rats

The usefulness of selective inhibitors of type 4 phosphodiesterase (PDE4) in the treatment of inflammation and pulmonary diseases is limited by their side effects: nausea and vomiting. We studied the effect of three structurally diverse PDE4 inhibitors on the vagal nerve afferent and efferent fibers in anesthetized rats. The effects of RS14203, (R)-rolipram, and CT-2450 were evaluated on the von Bezold-Jarisch reflex (vagal afferent fibers) and in a model of vagal electrical stimulation (vagal efferent fibers). All three PDE4 inhibitors were administered at 1, 10, or 100 microg/kg (iv) 15 min prior to the induction of bradycardia by an iv injection of 2-methyl-5-HT (von Bezold-Jarisch reflex) or by vagal electrical stimulation. At 100 microg/kg, RS14203 significantly potentiated the 2-methyl-5-HT response. No statistically significant effects were observed with (R)-rolipram or CT-2450 at the doses studied. RS14203, (R)-rolipram, or CT-2450 (1-100 microg/kg iv) did not affect the bradycardia induced by vagal electrical stimulation. Consequently, our results show that RS14203 selectively facilitates serotoninergic neurotransmission in vagal afferent fibers. The emetic action of RS14203 may be mediated by this mechanism.     

Capsaicin-sensitive vagal afferents and CCK in inhibition of gastric motor function induced by intestinal nutrients

The role of vagal afferent pathways and cholecystokinin (CCK) in mediating changes in gastric motor function after a meal was investigated in urethane-anesthetized rats. Proximal gastric motor function was measured manometrically, and nutrients were infused into an isolated segment of duodenum. Inhibition of gastric motility in response to duodenal infusion of protein (peptone or casein), but not carbohydrate (glucose), was significantly attenuated by administration of the CCK antagonist, L364,718. Selective ablation of vagal afferents by perineural treatment with the sensory neurotoxin, capsaicin, significantly reduced responses to both duodenal protein and glucose. These results suggest that protein in the duodenum decreases proximal gastric motor function via release of CCK and a vagal capsaicin-sensitive afferent pathway. In contrast, glucose acts via a capsaicin-sensitive vagal pathway not involving CCK. Thus separate neural and hormonal mechanisms mediate the effects of different nutrients in the duodenal feedback regulation of gastric motor function.     

Responses of efferent octopaminergic thoracic unpaired median neurons in the locust to visual and mechanosensory signals

Insect thoracic ganglia contain efferent octopaminergic unpaired median neurons (UM neurons) located in the midline, projecting bilaterally and modulating neuromuscular transmission, muscle contraction kinetics, sensory sensitivity and muscle metabolism. In locusts, these neurons are located dorsally or ventrally (DUM- or VUM-neurons) and divided into functionally different sub-populations activated during different motor tasks. This study addresses the responsiveness of locust thoracic DUM neurons to various sensory stimuli. Two classes of sense organs, cuticular exteroreceptor mechanosensilla (tactile hairs and campaniform sensilla), and photoreceptors (compound eyes and ocelli) elicited excitatory reflex responses. Chordotonal organ joint receptors caused no responses. The tympanal organ (Müller's organ) elicited weak excitatory responses most likely via generally increased network activity due to increased arousal. Vibratory stimuli to the hind leg subgenual organ never elicited responses. Whereas DUM neurons innervating wing muscles are not very responsive to sensory stimulation, those innervating leg and other muscles are very responsive to stimulation of exteroreceptors and hardly responsive to stimulation of proprioceptors. After cutting both cervical connectives all mechanosensory excitation is lost, even for sensory inputs from the abdomen. This suggests that, in contrast to motor neurons, the sensory inputs to octopaminergic efferent neuromodulatory cells are pre-processed in the suboesophageal ganglion.     

Water stimulation of the posterior oral cavity induces inhibition of gastric motility

The response of gastric motility to the administration of water and saline in the larynx and epiglottis was investigated in urethan-chloralose anesthetized rats. Administration of water inhibited motility of the distal stomach, but 0.15 M NaCl did not induce the inhibitory response. Bilateral sectioning of the superior laryngeal nerve (SLN) abolished the inhibitory response induced by water. Bilateral cervical vagotomies abolished the inhibitory responses, although spinal transection did not affect the inhibitory response. These inhibitory responses have been observed in immobilized animals. The degree of inhibition by water and hypotonic saline was negatively correlated with the sodium concentration. In contrast, the degree of inhibition to hypertonic saline was positively correlated with the sodium concentration. The proximal stomach also showed a reduction in intragastric pressure in response to the administration of water. These findings suggest that water-responsive afferent neurons in the SLN suppress gastric motility via the vagal efferent nerve.     

Capsaicin-sensitive vagal afferents contribute to gastric acid and vascular responses to intracisternal TRH analog

Central injection of TRH or its stable analog, RX77368, produces a vagal cholinergic stimulation of gastric acid secretion, mucosal blood flow and motor function. In the present study, we have investigated the contribution of capsaicin-sensitive vagal afferent fibers to the gastric responses to intracisternal injection of RX77368. Gastric acid secretion, measured in acute gastric fistula rats anesthetized with urethane, in response to intracisternal injection of RX77368 (3-30 ng) was reduced by 21-65% by perineural pretreatment of the vagus nerves with capsaicin 10-20 days before experiments. The increase in gastric mucosal blood flow measured by hydrogen gas clearance induced by intracisternal injection of RX77368 (30 ng) was also reduced by 65% in capsaicin-pretreated rats. In contrast, increases in gastric motor function measured manometrically or release of gastric luminal serotonin in response to intracisternal injection of RX77368 (3-30 ng) were unaltered by capsaicin pretreatment. The mechanism by which vagal afferent fibers contribute to the secretory and blood flow responses to the stable TRH analog is unclear at present, but it is possible that the decrease in gastric mucosal blood flow by lesion of capsaicin-sensitive vagal afferents limits the secretory response.     

Tachykinins as modulators of the micturition reflex in the central and peripheral nervous system

In the normal urinary bladder, tachykinins (TKs) are expressed in a population of bladder nociceptors that is sensitive to the excitatory and desensitizing effects of capsaicin (i.e., capsaicin-sensitive primary afferent neurons (CSPANs)). Several endobiotics or xenobiotics excite CSPANs and release TKs and other mediators at both the peripheral and spinal cord level. The peripheral release of TKs determines a set of responses (known as neurogenic inflammation) that includes vasodilatation, plasma protein extravasation, smooth muscle contraction and stimulation of afferent nerves. Following chronic inflammation, both immune cells and capsaicin-resistant sensory neurons can de novo express TKs: whether these pools of TKs are releasable and contribute to inflammatory processes is presently unsettled. At the spinal cord level, the release of TKs contributes in determining an altered pattern of vesicourethral reflexes in response to nociceptive stimulation of the bladder by conveying: (a) the afferent transmission to supraspinal sites, and (b) descending or sensory inputs to the sacral parasympathetic nucleus (SPN). Recent evidence also attribute a synergetic role of TKs in the supraspinal modulation of the sensory arm of the micturition reflex.The overall available information suggests that TK receptor antagonists may affect bladder motility/reflexes which occur during different pathological states, while having little influence on the normal motor bladder function.     

Modulation of the masseteric reflex by gastric vagal afferents

Several investigations have shown that the vagal nerve can affect the reflex responses of the masticatory muscles acting at level either of trigeminal motoneurons or of the mesencephalic trigeminal nucleus (MTN). The present experiments have been devoted to establish the origin of the vagal afferent fibres involved in modulating the masseteric reflex. In particular, the gastric vagal afferents were taken into consideration and selective stimulations of such fibres were performed in rabbit. Conditioning electrical stimulation of truncus vagalis ventralis (TVV) reduced the excitability of the MTN cells as shown by a decrease of the antidromic response recorded from the semilunar ganglion and elicited by MTN single-shock electrical stimulation. Sympathetic and cardiovascular influences were not involved in these responses. Mechanical stimulation of gastric receptors, by means of gastric distension, clearly diminished the amplitude of twitch tension of masseteric reflex and inhibited the discharge frequency of proprioceptive MTN units. The effect was phasic and depended upon the velocity of distension. Thus the sensory volleys originating from rapid adapting receptors reach the brain stem through vagal afferents and by means of a polysynaptic connection inhibits the masseteric reflex at level of MTN cells.     

Gastric ulcers reduce A-type potassium currents in rat gastric sensory ganglion neurons

Voltage-dependent potassium currents are important contributors to neuron excitability and thus also to hypersensitivity after tissue insult. We hypothesized that gastric ulcers would alter K(+) current properties in primary sensory neurons. The rat stomach was surgically exposed, and a retrograde tracer (1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine methanesulfonate) was injected into multiple sites in the stomach wall. Inflammation and ulcers were produced by 10 injections of 20% acetic acid (HAc) in the gastric wall. Saline (Sal) injections served as control. Nodose or T9-10 dorsal root ganglia (DRG) cells were harvested and cultured 7 days later to record whole cell K(+) currents. Gastric sensory neurons expressed transient and sustained outward currents. Gastric inflammation significantly decreased the A-type K(+) current density in DRG and nodose neurons (Sal vs. HAc-DRG: 82.9 +/- 7.9 vs. 46.5 +/- 6.1 pA/pF; nodose: 149.2 +/- 10.9 vs. 71.4 +/- 11.8 pA/pF), whereas the sustained current was not altered. In addition, there was a significant shift in the steady-state inactivation to more hyperpolarized potentials in nodose neurons (Sal vs. HAc: -76.3 +/- 1.0 vs. -83.6 +/- 2.2 mV) associated with an acceleration of inactivation kinetics. These data suggest that a reduction in K(+) currents contributes, in part, to increased neuron excitability that may lead to development of dyspeptic symptoms.     

Vagal modulation of intestinal afferent sensitivity to systemic LPS in the rat

The central nervous system modulates inflammation in the gastrointestinal tract via efferent vagal pathways. We hypothesized that these vagal efferents receive synaptic input from vagal afferents, representing an autonomic feedback mechanism. The consequence of this vagovagal reflex for afferent signal generation in response to LPS was examined in the present study. Different modifications of the vagal innervation or sham procedures were performed in anesthetized rats. Extracellular mesenteric afferent nerve discharge and systemic blood pressure were recorded in vivo before and after systemic administration of LPS (6 mg/kg iv). Mesenteric afferent nerve discharge increased dramatically following LPS, which was unchanged when vagal efferent traffic was eliminated by acute vagotomy. In chronically vagotomized animals, to eliminate both vagal afferent and efferent traffic, the increase in afferent firing 3.5 min after LPS was reduced to 3.2 +/- 2.5 impulses/s above baseline compared with 42.2 +/- 2.0 impulses/s in controls (P < 0.001). A similar effect was observed following perivagal capsaicin, which was used to eliminate vagal afferent traffic only. LPS also caused a transient hypotension (<10 min), a partial recovery, and then persistent hypertension that was exacerbated by all three procedures. Mechanosensitivity was increased 15 min following LPS but had recovered at 30 min in all subgroups except for the chronic vagotomy group. In conclusion, discharge in capsaicin-sensitive mesenteric vagal afferents is augmented following systemic LPS. This activity, through a vagovagal pathway, helps to attenuate the effects of septic shock. The persistent hypersensitivity to mechanical stimulation after chronic vagal denervation suggests that the vagus exerts a regulatory influence on spinal afferent sensitization following LPS.     

Inhibitory effects of beta-alanine on the vagal efferent and afferent excitatory responses of the stomach to stimulation of vagal trunk in cats.

The effects of beta-alanine on the electrically evoked vagal efferent (hexamethonium-sensitive initial excitatory response) and afferent (hexamethonium-resistant delayed excitatory response) responses of the cat stomach were studied. beta-alanine (30 to 300 micrograms/kg, i.v.) dose-dependently inhibited both the efferent and afferent response. The IC50 values of beta-alanine on the efferent and afferent response were 296 +/- 65 micrograms/kg and 128 +/- 35 microgram/kg, respectively. Maximal inhibitory effects of beta-alanine (300 micrograms/kg, i.v.) appeared about 1 hr after the injection. Glycine and taurine (100 to 10,000 micrograms/kg) did not affect these responses. Treatment with hexamethonium (10 mg/kg, i.v.) prevented the efferent response, but augmented the afferent response. The treatment with hexamethonium abolished the inhibitory effect of beta-alanine on the afferent response. Both picrotoxin (100 and 500 micrograms/kg, i.v.) and bicuculline (2000 micrograms/kg, i.v.) antagonized the inhibitory effects of beta-alanine on the vagal efferent and afferent responses of the stomach. The present experiments clearly demonstrated that beta-alanine inhibited both the vagal efferent and afferent excitatory responses of stomach to electrical stimulation of vagal trunk in cats.     

Results of simulated mastication suggest existence of a periodontogastric motility reflex

Various reflexes inhibit gastric motor activity. Might a contrary one permit the oral region to increase gastric motility? Ten fasted rats were allowed to feed for 15 min. Following anesthesia and cannula insertion, antral pressure was recorded during three consecutive 5-min intervals: the baseline, procedure, and postprocedure periods. The procedure involved manually lowering and raising the mandible about once per second, causing repetitive molar occlusion. Doing this when food is in the stomach resembles conditions as the latter part of a meal is consumed. Gastric motor events increased from 1.10 +/- 1.67 (mean +/- SD) to 5.50 +/- 4.12 per 5 min during the procedure (p < 0.05) and 5.80 +/- 3.97 in the ensuing period (p < 0.05). The findings suggest an excitatory reflex following stimulation of mechanoreceptors in one or more sites related to mastication: the periodontium, temporomandibular joints, or masticatory muscles. Because rubbing the maxillary molars while the mouth remained constantly open also increased motor events, the periodontium is the most likely location of the receptors. They and associated trigeminal neurons would comprise the reflex's afferent arm. The vagi, perhaps with intermediaries, are its likely efferent arm. In these recently fed rats this reflex acts despite receptive relaxation and enterogastric reflexes to increase distal gastric motor activity.     

Role of bradykinin in gastric vasodilation caused by hypertonic saline and acid back diffusion

Protective vasodilation in response to tissue injury and acid back diffusion is associated with release of bradykinin in the rat stomach. We hypothesized that bradykinin might be involved in mechanisms behind such vasodilation via influence on mast cells and sensory neurons. Acid back diffusion after mucosal barrier disruption with hypertonic saline evoked degranulation of mast cells in the rat stomach wall. Acid back diffusion was also associated with increased luminal release of histamine and gastric blood flow in normal rats, but not in mast cell-deficient rats. Bradykinin (BK(2)) receptor blockade inhibited degranulation of submucosal mast cells in the stomach and attenuated gastric vasodilation both in response to acid back diffusion and after stimulation of sensory neurons with capsaicin. Gastric vasodilation caused by mucosal injury with hypertonic saline alone was associated with degranulation of mucosal mast cells. These events were unaffected by inhibition of prostaglandin synthesis, whereas bradykinin (BK(2)) receptor blockade was associated with abolished vasodilation and inhibition of mucosal mast cell degranulation. We conclude that bradykinin is involved in gastric vasodilation caused by hypertonic injury alone via influence on mast cells, and by acid back diffusion via influence on both sensory neurons and mast cells.     

Functional vagal input to gastric myenteric plexus as assessed by vagal stimulation-induced Fos expression

Immunohistochemical detection of c-Fos expression was used to identify gastric myenteric plexus neurons that receive excitatory input from vagal efferent neurons activated by electrical stimulation of the cervical vagi in anesthetized rats. Vagal stimulation-induced Fos expression increased with higher pulse frequency, so that with 16 Hz (rectangular pulses of 1 mA/0.5 ms for 30 min) approximately 30% and with 48 Hz 90% of all neurons near the lesser curvature were Fos positive. In sham-stimulated rats there was no Fos expression. The percentage of Fos-activated neurons was only slightly smaller (85% with 48 Hz) near the greater curvature. Prior atropine administration (1 mg/kg ip) had little effect on vagal stimulation-induced Fos expression, and in unilaterally stimulated rats there was no Fos expression on the contralateral (noninnervated) side of the stomach, ruling out mediation by gastric motility or secretory responses. However, polysynaptic recruitment of third- and higher-order neurons cannot be ruled out completely. These results support the idea that, at least in the stomach, functional excitatory innervation of myenteric plexus neurons by the efferent vagus is profuse and widespread, refuting the notion of only a few vagal "command neurons."     

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.Abbreviations BSA Bovine serum albumin - CGRP calcitonin generelated peptide - DiA carbocyanine dye A - DiI carbocyanine dye I - dmnX dorsal motor nucleus of vagus - DMSO dimethylsulfoxide - ENK enkephalin - FITC fluorescin isothiocyanate - NADPH diaphorase nicotinamide adenine diphosphate - NPY neuropeptide Y - NTS nucleus tractus solitarii - PBS phosphate-buffered saline - VIP vasoactive intestinal peptide - WGA-HRP wheat-germ agglutinine-horseradish peroxidase     

Laryngeal motor control in frogs: Role of vagal and laryngeal feedback

Using decerebrate frogs (Rana catesbeiana), we investigated the role of vagal and laryngeal sensory feedback in controlling motor activation of the larynx. Vagal and laryngeal nerve afferents were activated by electrical stimulation of the intact vagal and laryngeal nerves. Pulmonary afferents were activated by lung inflation. Reflex responses were recorded by measuring efferent activity in the laryngeal branch of the vagus (Xℓ) and changes in glottal aperture. Two glottic closure reflexes were identified, one evoked by lung inflation or electrical stimulation of the main branch of the vagus (Xm), and the other by electrical stimulation of Xℓ. Lung inflation evoked a decrementing burst of Xℓ efferent activity and electrical stimulation of Xm resulted in a brief burst of Xℓ action potentials. Electrical stimulation of Xℓ evoked a triphasic mechanical response, an abrupt glottal constriction followed by glottal dilatation followed by a long-lasting glottal constriction. The first phase was inferred to be a direct (nonreflex) response to the stimulus, whereas the second and third represent reflex responses to the activation of laryngeal afferents. Intracellular recordings of membrane potential of vagal motoneurons of lung and nonlung types revealed EPSPs in both types of neurons evoked by stimulation of Xm or Xℓ, indicating activation of glottal dilator and constrictor motoneurons. In summary, we have identified two novel reflexes producing glottic closure, one stimulated by activation of pulmonary receptors and the other by laryngeal receptors. The former may be part of an inspiratory terminating reflex and the latter may represent an airway protective reflex. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 213–222, 1997