Catecholaminergic microcircuitry controlling the output of airway-related vagal preganglionic neurons.

In this study, we have investigated the ultrastructure and function of the catecholaminergic circuitry modulating the output of airway-related vagal preganglionic neurons (AVPNs) in ferrets. Immunoelectron microscopy was employed to characterize the nature of catecholaminergic innervation of AVPN at the ultrastructural level. In addition, immunofluorescence was used to examine the expression of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) on AVPNs, and norepinephrine release within the rostral nucleus ambiguous (rNA) was measured by using microdialysis. Physiological experiments were performed to determine the effects of stimulation of the noradrenergic locus coeruleus (LC) cell group on airway smooth muscle tone. The results showed that 1) catecholaminergic nerve endings terminate in the vicinity of identified AVPNs but very rarely form axosomatic or axodendritic synapses with the AVPNs that innervate the extrathoracic trachea; 2) AVPNs express the alpha(2A)-AR; 3) LC stimulation-induced norepinephrine release within the rNA region was associated with airway smooth muscle relaxation; and 4) blockade of alpha(2A)-AR on AVPNs diminished the inhibitory effects of LC stimulation on airway smooth muscle tone. It is concluded that a noradrenergic circuit originating within the LC is involved in the regulation of AVPN activity within the rNA, and stimulation of the LC dilates the airways by the release of norepinephrine and activation of alpha(2A)-AR expressed by AVPNs, mainly via volume transmission.     

A GABAergic inhibitory microcircuit controlling cholinergic outflow to the airways.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.     

Activation of the midbrain periaqueductal gray induces airway smooth muscle relaxation.

In this study, we examined effects of chemical stimulation of the ventrolateral region of the midbrain periaqueductal gray (vl PAG) on airway smooth muscle tone. We observed that in anesthetized, paralyzed, and artificially ventilated ferrets, vl PAG stimulation elicited airway smooth muscle relaxation. To clarify the mechanisms underlying this observation, we examined the GABA-GABA(A) receptor signaling pathway by 1) examining the expression of GABA(A) receptors on airway-related vagal preganglionic neurons (AVPNs) located in the rostral nucleus ambiguus region (rNA), by use of receptor immunochemistry and confocal microscopy; 2) measuring GABA release within the rNA by using microdialysis; and 3) performing physiological experiments to determine the effects of selective blockade of GABA(A) receptors expressed by AVPNs in the rNA region on vl PAG-induced airway relaxation, thereby defining the role of the GABA(A) receptor subtype in this process. We observed that AVPNs located in the rNA region do express the GABA(A) receptor beta-subtype. In addition, we demonstrated that activation of vl PAG induced GABA release within the rNA region, and this release was associated with airway smooth muscle relaxation. Blockade of the GABA(A) receptor subtype expressed by AVPNs in the rNA by bicuculline diminished the inhibitory effects of vl PAG stimulation on airway smooth muscle tone. These data indicate, for the first time, that activation of vl PAG dilates the airways by a release of GABA and activation of GABA(A) receptors expressed by AVPNs.     

Allergic lung inflammation affects central noradrenergic control of cholinergic outflow to the airways in ferrets.

Brain stem noradrenergic cell groups mediating autonomic responses to stress project to airway-related vagal preganglionic neurons (AVPNs). In ferrets, their activation produces withdrawal of cholinergic outflow to the airways via release of norepinephrine and activation of alpha(2A)-adrenergic receptors (alpha(2A)-AR) expressed by AVPNs. In these studies, we examined the effects of allergen exposure of the airway (AE) with ovalbumin on noradrenergic transmission regulating the activity of AVPNs and, consequently, airway smooth muscle tone. Experiments were performed in vehicle control (Con) and AE ferrets. Microperfusion of an alpha(2A)-AR agonist (guanabenz) in close proximity to AVPNs elicited more pronounced effects in Con than AE ferrets, including a decrease in unit activity and reflexly evoked responses of putative AVPN neurons with a corresponding decrease in cholinergic outflow to the airways. Although no differences were found in the extent of noradrenergic innervation of the AVPNs, RT-PCR and Western blot studies demonstrated that AE and repeated exposure to antigen significantly reduced expression of alpha(2A)-ARs at message and protein levels. These findings indicate that, in an animal model of allergic asthma, sensitization and repeated challenges with a specific allergen diminish central inhibitory noradrenergic modulation of AVPNs, possibly via downregulation of alpha(2A)-AR expression by these neurons.     

Absence of nonadrenergic noncholinergic relaxation in the cat cervical trachea

Published in vivo experiments have not supported in vitro reports of the presence of nonadrenergic noncholinergic (NANC) inhibitory pathways in the cat trachea. We therefore examined these pathways, measuring tension in an innervated tracheal segment, flow resistance in more distal airways, and dynamic compliance, in 10 anesthetized mechanically ventilated cats. Initially, cervical vagal stimulation evoked contraction followed by relaxation of smooth muscle of trachea and lower airways; sympathetic stimulation evoked relaxation only. After muscarinic blockade and restoration of smooth muscle tone with 5-hydroxytryptamine (5-HT) applied topically to the tracheal mucosa, vagal stimulation did not affect tracheal segment tension, whereas sympathetic-evoked relaxation was preserved. Similar results were found when tone was restored with intravenous 5-HT, with vagal stimulation also decreasing resistance and increasing compliance. We conclude that NANC pathways are present in lower airways but not in the cervical trachea of the cat. We hypothesize that parasympathetic constriction of cat airway smooth muscle can occur without simultaneous NANC activation, whereas NANC activity occurs only in tandem with parasympathetic stimulation.     

Brain stem excitatory and inhibitory signaling pathways regulating bronchoconstrictive responses.

This review summarizes recent work on two basic processes of central nervous system (CNS) control of cholinergic outflow to the airways: 1) transmission of bronchoconstrictive signals from the airways to the airway-related vagal preganglionic neurons (AVPNs) and 2) regulation of AVPN responses to excitatory inputs by central GABAergic inhibitory pathways. In addition, the autocrine-paracrine modulation of AVPNs is briefly discussed. CNS influences on the tracheobronchopulmonary system are transmitted via AVPNs, whose discharge depends on the balance between excitatory and inhibitory impulses that they receive. Alterations in this equilibrium may lead to dramatic functional changes. Recent findings indicate that excitatory signals arising from bronchopulmonary afferents and/or the peripheral chemosensory system activate second-order neurons within the nucleus of the solitary tract (NTS), via a glutamate-AMPA signaling pathway. These neurons, using the same neurotransmitter-receptor unit, transmit information to the AVPNs, which in turn convey the central command to airway effector organs: smooth muscle, submucosal secretory glands, and the vasculature, through intramural ganglionic neurons. The strength and duration of reflex-induced bronchoconstriction is modulated by GABAergic-inhibitory inputs and autocrine-paracrine controlling mechanisms. Downregulation of GABAergic inhibitory influences may result in a shift from inhibitory to excitatory drive that may lead to increased excitability of AVPNs, heightened airway responsiveness, and sustained narrowing of the airways. Hence a better understanding of these normal and altered central neural circuits and mechanisms could potentially improve the design of therapeutic interventions and the treatment of airway obstructive diseases.     

Catecholamines abolish vagal but not acetylcholine tone in the intact cat trachea

To obtain evidence in the airways that catecholamines inhibit cholinergic neurotransmission, we recorded transverse tension in the posterior wall of an upper tracheal segment in anesthetized cats and compared the inhibitory effect of stimulating cervical sympathetic nerves when segment contraction was evoked by endogenous acetylcholine (vagal tone) with the effect when contraction was evoked by exogenous acetylcholine applied directly to the mucosal surface of the tracheal segment (ACh tone). We found that sympathetic stimulation abolished all contraction evoked by vagal tone but reduced ACh tone by only one-half. In a second group of cats we compared the inhibitory effects of sympathetic stimulation and intravenous isoproterenol during vagal and ACh tone and also during tone evoked by exogenous 5-hydroxytryptamine (5-HT tone). Sympathetic stimulation or isoproterenol injection abolished all vagal and 5-HT tone but again reduced ACh tone by only one-half. Our results suggest that catecholamines released from sympathetic nerves or injected into the circulation completely inhibit vagal tone. This inhibition may be partially responsible for inducing relaxation in airway smooth muscle.     

Musings on the wanderer: what's new in our understanding of vago-vagal reflex? IV. Current concepts of vagal efferent projections to the gut

Vagal efferents, consisting of distinct lower motor and preganglionic parasympathetic fibers, constitute the motor limb of vagally mediated reflexes. Arising from the nucleus ambiguus, vagal lower motor neurons (LMN) mediate reflexes involving striated muscles of the orad gut. LMNs provide cholinergic innervation to motor end plates that are inhibited by myenteric nitrergic neurons. Preganglionic neurons from the dorsal motor nucleus implement parasympathetic motor and secretory functions. Cholinergic preganglionic neurons form parallel inhibitory and excitatory vagal pathways to smooth muscle viscera and stimulate postganglionic neurons via nicotinic and muscarinic receptors. In turn, the postganglionic inhibitory neurons release ATP, VIP, and NO, whereas the excitatory neurons release ACh and substance P. Vagal motor effects are dependent on the viscera's intrinsic motor activity and the interaction between the inhibitory and excitatory vagal influences. These interactions help to explain the physiology of esophageal peristalsis, gastric motility, lower esophageal sphincter, and pyloric sphincter. Vagal secretory pathways are predominantly excitatory and involve ACh and VIP as the postganglionic excitatory neurotransmitters. Vagal effects on secretory functions are exerted either directly or via release of local mediators or circulating hormones.     

Acid-Sensing Ion Channel 1a Contributes to Airway Hyperreactivity in Mice

Neurons innervating the airways contribute to airway hyperreactivity (AHR), a hallmark feature of asthma. Several observations suggested that acid-sensing ion channels (ASICs), neuronal cation channels activated by protons, might contribute to AHR. For example, ASICs are found in vagal sensory neurons that innervate airways, and asthmatic airways can become acidic. Moreover, airway acidification activates ASIC currents and depolarizes neurons innervating airways. We found ASIC1a protein in vagal ganglia neurons, but not airway epithelium or smooth muscle. We induced AHR by sensitizing mice to ovalbumin and found that ASIC1a^-/- mice failed to exhibit AHR despite a robust inflammatory response. Loss of ASIC1a also decreased bronchoalveolar lavage fluid levels of substance P, a sensory neuropeptide secreted from vagal sensory neurons that contributes to AHR. These findings suggest that ASIC1a is an important mediator of AHR and raise the possibility that inhibiting ASIC channels might be beneficial in asthma.     

Vagal control of central and peripheral pulmonary resistance in developing piglets

To study the postnatal maturation of vagal control of airway muscle tone, we determined the effects of vagotomy and supramaximal vagal stimulation on the resistance of the respiratory system in eight newborn and seven 6-wk-old piglets. Because the lung periphery has distinctive responses to cholinergic agonists and a lower density of vagal fibers and cholinergic receptors than the central airways, we partitioned the respiratory resistance of the piglets between central airways (Rc) and peripheral airways and lung tissue (Rp) with bronchial catheters inserted in a retrograde manner. The piglets were anesthetized with alpha-chloralose and ventilated with positive airway pressure. Vagotomy did not change Rc or Rp in either the newborn or the 6-wk-old piglets. Vagal stimulation, on the other hand, increased both Rc (median increase 53% in the newborn and 72% in the 6-wk-old piglets) and Rp (54 and 42%, respectively). At all states of vagal tone, Rp increased as the lungs were inflated, suggesting a large contribution of tissue viscoelasticity to this resistance. Our results demonstrate that vagal bronchomotor tone is absent during mechanical ventilation with positive pressure in the developing piglet. However, vagal innervation of both central airways and tissue contractile elements is functionally competent at the time of birth in this species.     

Expression of mechanogated two-pore domain potassium channels in mouse lungs: special reference to mechanosensory airway receptors

Afferent activities arising from sensory nerve terminals located in lungs and airways are carried almost exclusively by fibres travelling through the vagus nerve. Based on electrophysiological investigations, intrapulmonary airway-related vagal afferent receptors have been classified into three main subtypes, two of which are myelinated and mechanosensitive, i.e., rapidly and slowly adapting receptors. To allow for a full functional identification of the distinct populations of airway receptors, morphological and neurochemical characteristics still need to be determined. Nerve terminals visualised using markers for myelinated vagal afferents seem to be almost uniquely associated with two morphologically well-formed airway receptor end organs, smooth muscle-associated airway receptors (SMARs) and neuroepithelial bodies (NEBs), localised in airway smooth muscle and epithelium, respectively. Due to the lack of a selective marker for SMARs in mice, no further neurochemical coding is available today. NEBs are extensively innervated diffusely spread groups of neuroendocrine cells in the airway epithelium, and are known to receive at least two separate populations of myelinated vagal afferent nerve terminals. So far, however, no evidence has been reported for the expression of channels that may underlie direct sensing and transduction of mechanical stimuli by the receptor terminals in NEBs and SMARs. This study focused on the expression of mechanogated two-pore domain K⁺ (K_2P) channels, TREK-1 and TRAAK, in mouse airways and more particular in the NEB micro-environment and in SMARs by multiple immunostaining. TREK-1 could be detected on smooth muscle cells surrounding intrapulmonary airways and blood vessels, while TRAAK was expressed on myelinated vagal afferents terminating both in SMARs and in the NEB micro-environment. Co-stainings with known markers for subpopulations of myelinated vagal afferents and general neuronal markers revealed that all identified SMARs exhibit TRAAK immunoreactivity, and that at least three subpopulations exist in mouse airways. Also, the intraepithelial terminals of both subpopulations of NEB-associated myelinated vagal sensory nerve fibres were shown to express TRAAK. In conclusion, the present study finally characterised an intrinsically mechanosensitive ion channel, the K_2P channel TRAAK, on the terminals of identified myelinated vagal nodose airway afferents, organised as SMARs and as components of the innervation of NEBs. These data support the hypothesis that both SMARs and NEBs harbour the morphological counterparts of electrophysiologically identified myelinated vagal airway mechanoreceptors. TRAAK appears to be strongly involved in regulating airway mechanosensing since it was found to be expressed on the terminals of all subpopulations of potential vagal mechanosensors.     

Reflex regulation of airway smooth muscle tone.

Autonomic nerves in most mammalian species mediate both contractions and relaxations of airway smooth muscle. Cholinergic-parasympathetic nerves mediate contractions, whereas adrenergic-sympathetic and/or noncholinergic parasympathetic nerves mediate relaxations. Sympathetic-adrenergic innervation of human airway smooth muscle is sparse or nonexistent based on histological analyses and plays little or no role in regulating airway caliber. Rather, in humans and in many other species, postganglionic noncholinergic parasympathetic nerves provide the only relaxant innervation of airway smooth muscle. These noncholinergic nerves are anatomically and physiologically distinct from the postganglionic cholinergic parasympathetic nerves and differentially regulated by reflexes. Although bronchopulmonary vagal afferent nerves provide the primary afferent input regulating airway autonomic nerve activity, extrapulmonary afferent nerves, both vagal and nonvagal, can also reflexively regulate autonomic tone in airway smooth muscle. Reflexes result in either an enhanced activity in one or more of the autonomic efferent pathways, or a withdrawal of baseline cholinergic tone. These parallel excitatory and inhibitory afferent and efferent pathways add complexity to autonomic control of airway caliber. Dysfunction or dysregulation of these afferent and efferent nerves likely contributes to the pathogenesis of obstructive airways diseases and may account for the pulmonary symptoms associated with extrapulmonary disorders, including gastroesophageal reflux disease, cardiovascular disease, and rhinosinusitis.     

Reciprocal activity of longitudinal and circular muscle during intestinal peristaltic reflex

A two-compartment, flat-sheet preparation of rat colon was devised, which enabled exclusive measurement of longitudinal muscle activity during the ascending and descending phases of the peristaltic reflex. A previous study using longitudinal muscle strips revealed the operation of an integrated neuronal circuit consisting of somatostatin, opioid, and VIP/pituitary adenylate cyclase-activating peptide (PACAP)/nitric oxide synthase (NOS) interneurons coupled to cholinergic/tachykinin motor neurons innervating longitudinal muscle strips that could lead to descending contraction and ascending relaxation of this muscle layer. Previous studies in peristaltic preparations have also shown that an increase in somatostatin release during the descending phase causes a decrease in Met-enkephalin release and suppression of the inhibitory effect of Met-enkephalin on VIP/PACAP/NOS motor neurons innervating circular muscle and a distinct set of VIP/PACAP/NOS interneurons. The present study showed that in contrast to circular muscle, longitudinal muscle contracted during the descending phase and relaxed during the ascending phase. Somatostatin antiserum inhibited descending contraction and augmented ascending relaxation of longitudinal muscle, whereas naloxone had the opposite effect. VIP and PACAP antagonists inhibited descending contraction of longitudinal muscle and augmented ascending relaxation. Atropine and tachykinin antagonists inhibited descending contraction of longitudinal muscle. As shown in earlier studies, the same antagonists and antisera produced opposite effects on circular muscle. We conclude that longitudinal muscle contracts and relaxes in reverse fashion to circular muscle during the peristaltic reflex. Longitudinal muscle activity is regulated by excitatory VIP/PACAP/NOS interneurons coupled to cholinergic/tachykinin motor neurons innervating longitudinal muscle.     

Neuro-Anatomical Evidence Indicating Indirect Modulation of Macrophages by Vagal Efferents in the Intestine but Not in the Spleen

Background

Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine.

Methods

Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages.

Results

Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers.

Conclusion

In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages.     

Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.     

Pituitary adenylate cyclase activating peptide (PACAP) in guinea-pig lung: distribution and dilatory effects.

The lower airways of guinea-pigs were analyzed for pituitary adenylate cyclase activating peptide (PACAP) using immunocytochemistry. In the trachea a moderate supply of PACAP-immunoreactive nerve fibers occurred around smooth muscle bundles, glands and small blood vessels. In the lung, PACAP-immunoreactive nerve fibers were distributed around small glands and bronchi. A rich supply of PACAP immunoreactive nerve fibers was found around blood vessels in the lungs. PACAP-suppressed smooth muscle responses were analysed using isolated circular segments of trachea, pulmonary arteries and aorta of guinea-pigs. In both airways and arteries PACAP caused a concentration-dependent relaxation of precontracted segments. The maximal relaxation effects were more pronounced in the airways than in the arteries while the order of potency was aorta greater than pulmonary artery greater than trachea. The effect of PACAP was compared to those of acetylcholine (ACh) and vasoactive intestinal peptide (VIP). In the pulmonary artery the vasomotor responses expressed as maximal dilatation had the order: ACh greater than VIP = PACAP while the order of potency was PACAP = VIP greater than ACh. In the trachea, PACAP was slightly more potent than VIP. The relaxatory responses to PACAP in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine, cimetidine and Spantide. Removal of the endothelium abolished PACAP-induced vascular relaxation. Conceivably, PACAP-containing nerve fibers play a role in the regulation of airway resistance and local blood flow.     

Multiple mechanisms of reflex bronchospasm in guinea pigs.

The mechanisms of histamine- and bradykinin-induced reflex bronchospasm were determined in anesthetized guinea pigs. With intravenous administration, both autacoids evoked dose-dependent increases in tracheal cholinergic tone. Vagotomy or atropine prevented these tracheal reflexes. When delivered as an aerosol, bradykinin readily increased tracheal cholinergic tone, whereas histamine aerosols were much less effective at inducing tracheal reflexes. Also, unlike histamine, bradykinin could evoke profound increases in cholinergic tone without directly or indirectly (e.g., prostanoid dependent) inducing measurable airway smooth muscle contraction resulting in bronchospasm. Neither autacoid required de novo synthesis of prostanoids or nitric oxide to induce reflex tracheal contractions. Combined cyclooxygenase inhibition and tachykinin-receptor antagonism did, however, abolish all effects of bradykinin in the airways, whereas responses to histamine were unaffected by these pretreatments. The data indicate that histamine and bradykinin initiate reflex bronchospasm by differential activation of vagal afferent nerve subtypes. We speculate that selective activation of either airway C fibers or airway rapid adapting receptors can initiate reflex bronchospasm.     

Role of endogenous nitric oxide in hyperoxia-induced airway hyperreactivity in maturing rats.

We sought to define the effects of maturation and hyperoxic stress on nitric oxide (NO)-induced modulation of bronchopulmonary responses to stimulation of vagal preganglionic nerve fibers. Experiments were performed on decerebrate, paralyzed, and ventilated rat pups at 6-7 days (n = 21) and 13-15 days of age (n = 23) breathing room air and on rat pups 13-15 days of age (n = 19) after exposure to hyperoxia (>/=95% inspired O(2) fraction for 4-6 days). Total lung resistance (RL) and lung elastance (EL) were measured by body plethysmograph. Vagal stimulation and release of acetylcholine caused a frequency-dependent increase in RL and EL in all animals. The RL response was significantly potentiated in normoxic animals by prior blockade of nitric oxide synthase (NOS) (P < 0.05). Hyperoxic exposure increased responses of RL to vagal stimulation (P < 0.05); however, after hyperoxic exposure, the potentiation of contractile responses by NOS blockade was abolished. The response of EL was potentiated by NOS blockade in the 13- to 15-day-old animals after both normoxic and hyperoxic exposure (P < 0.01). Morphometry revealed no effect of hyperoxic exposure on airway smooth muscle thickness. We conclude that NO released by stimulation of vagal preganglionic fibers modulates bronchopulmonary contractile responses to endogenously released acetylcholine in rat pups. Loss of this modulatory effect of NO could contribute to airway hyperreactivity after prolonged hyperoxic exposure, as may occur in bronchopulmonary dysplasia.     

Influence of ventrolateral surface of medulla on reflex tracheal constriction

To assess the role of structures located superficially near the ventrolateral surface of the medulla on the reflex constriction of tracheal smooth muscle that occurs when airway and pulmonary receptors are stimulated mechanically or chemically, experiments were conducted in alpha-chloralose-anesthetized, paralyzed, and artificially ventilated cats. Pressure changes within a bypassed segment of the trachea were used as an index of alterations smooth muscle tone. The effects of focal cooling of the intermediate areas or topically applied lidocaine on the ventral surface of the medulla on the response of the trachea to mechanical and chemical stimulation of airway receptors were examined. Atropine abolished tracheal constriction induced by mechanical stimulation of the carina or aerosolized histamine, showing that the responses were mediated over vagal pathways. Moderate cooling of the intermediate area (20 degrees C) or local application of lidocaine significantly decreased the tracheal constrictive response to mechanical activation of airway receptors. Furthermore, when the trachea was constricted by histamine, cooling of the intermediate area significantly diminished the increased tracheal tone, whereas rewarming restored tracheal tone to the previous level. These findings suggest that under the conditions of the experiments the ventral surface of the medulla plays an important role in constriction of the trachea by inputs from intrapulmonary receptors and in the modulation of parasympathetic outflow to airway smooth muscle.     

Different subpopulations of cholinergic and nitrergic myenteric neurones project to mucosa and circular muscle of the guinea-pig gastric fundus

Since the stomach lacks a well-developed ganglionated submucous plexus, the somata of enteric neurones innervating the muscle or the mucosa have to be localised within the myenteric plexus. The aim of this study was to determine the projection pathways and the neurochemical coding of myenteric neurones innervating these different targets in the gastric fundus. Myenteric cell bodies projecting to the mucosa or the circular muscle were retrogradely labelled by mucosa or muscle application of the fluorescent tracer DiI and subsequently characterised by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and/or neuropeptide Y (NPY). On average 143±91 and 89±49 myenteric neurones were labelled from the mucosa and the circular muscle, respectively. DiI-labelled neurones were either ChAT- or NOS-positive. DiI-labelled ChAT-positive neurones were mainly ascending and outnumbered NOS-positive neurones, which were mainly descending (79.3±6.2% vs 20.7±6.2% for mucosa neurones; 69.3±11.1% vs 30.7±11.1% for muscle neurones). Three ChAT-positive subpopulations (ChAT/–, ChAT/SP, ChAT/NPY) and two NOS-positive subpopulations (NOS/–, NOS/NPY) were found. ChAT/SP neurones projected mainly to the circular muscle (36.1±11.9% of the cholinergic muscle neurones; mucosa projection: 8.0±2.1%), whereas ChAT/NPY neurones projected mainly to the mucosa (38.1±9.2% of the cholinergic mucosa neurones; muscle projection: 5.7±2.4%). NOS/– cells projected predominantly to the muscle. This study demonstrates polarised pathways in the myenteric plexus consisting of ascending ChAT and descending NOS cells that innervate the circular muscle and the mucosa of the gastric fundus. The ChAT/SP neurones might function as circular muscle motor neurones, whereas ChAT/NPY neurones might represent secretomotor neurones.