Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors.

Vagal afferent fibres innervating thoracic structures such as the respiratory tract and oesophagus are diverse, comprising several subtypes of functionally distinct C-fibres and A-fibres. Both morphological and functional studies of these nerve subtypes would be advanced by selective, effective and long-term transduction of vagal afferent neurons with viral vectors. Here we addressed the hypothesis that vagal sensory neurons can be transduced with adeno-associated virus (AAV) vectors in vivo, in a manner that would be useful for morphological assessment of nerve terminals, using enhanced green fluorescent protein (eGFP), as well as for the selective knock-down of specific genes of interest in a tissue-selective manner. We found that a direct microinjection of AAV vectors into the vagal nodose ganglia in vivo leads to selective, effective and long-lasting transduction of the vast majority of primary sensory vagal neurons without transduction of parasympathetic efferent neurons. The transduction of vagal neurons by pseudoserotype AAV2/8 vectors in vivo is sufficiently efficient such that it can be used to functionally silence TRPV1 gene expression using short hairpin RNA (shRNA). The eGFP encoded by AAV vectors is robustly transported to both the central and peripheral terminals of transduced vagal afferent neurons allowing for bright imaging of the nerve endings in living tissues and suitable for structure-function studies of vagal afferent nerve endings. Finally, the AAV2/8 vectors are efficiently taken up by the vagal nerve terminals in the visceral tissue and retrogradely transported to the cell body, allowing for tissue-specific transduction.

Lysophosphatidic acid is detectable in human bronchoalveolar lavage fluids at baseline and increased after segmental allergen challenge.

BACKGROUND: Lysophosphatidic acid (LPA) is a biologically active lysophospholipid and a component of normal plasma. LPA binds to receptors expressed on circulating and structural lung cells and affects cell growth and activation. Whether LPA is present in the lung has not been previously reported. OBJECTIVE: To develop an assay to measure LPA in bronchoalveolar lavage (BAL) fluids, and to study the association between LPA and allergic airway inflammation. METHODS: Seventeen allergic subjects underwent bronchoscopy and segmental allergen challenge, followed 18 h later by BAL. Supernatants were analysed for LPA content using liquid chromatography and mass spectroscopy. Expression of LPA receptors on primary bronchial epithelial cells was analysed by immunolabelling, and the effects of LPA on epithelial cell barrier function was investigated by measuring transepithelial resistance. RESULTS: LPA was detectable in BAL from control lung segments, and significantly increased 18 h after allergen challenge. Polyunsaturated species of LPA were especially increased following segmental allergen challenge. LPA levels did not strongly correlate with the number or percentages of eosinophils, neutrophils of lymphocytes, whereas MIP-3alpha (CCL20) levels correlated significantly with the allergen-driven influx of lymphocytes. The levels of LPA from control sites correlated inversely with BAL protein content, suggesting that LPA promoted epithelial barrier integrity at baseline. Experiments using primary human bronchial epithelial cells confirmed that LPA tightened the epithelial cell barrier. CONCLUSION: Lysophosphatidic acid is detectable in human BAL fluids at baseline and its expression increases during allergic inflammation. LPA does not appear to be a dominant chemoattractant for eosinophils or lymphocytes during allergic airway inflammation. In the absence of ongoing inflammation, LPA may promote epithelial barrier integrity.

Exploring the stereochemical requirements for protease inhibition by ureidopeptides.

A novel 'ureidopeptide' substrate analog inhibitor of the HIV-1 protease, created by substitution of a urea for the scissile amide bond of a hexapeptide substrate, was synthesized and tested for inhibition of HIV-1 protease. This inhibitor was designed as a stereochemical mutant of an earlier ureidopeptide inhibitor in which the P1' phenylalanine residue was changed from an l-isomer to a d-isomer. This was done in an attempt to increase binding to the enzyme by compensating for a lengthening of the peptide backbone. The inhibitor was synthesized from two protected tripeptide precursors using an oxidative Hoffmann rearrangement of a C-terminal peptide amide. The new inhibitor was found to inhibit HIV-1 protease with an observed IC(50) of 47 mum.

Subtypes of vagal afferent C-fibres in guinea-pig lungs.

An ex vivo, vagally innervated, lung preparation was used to address the hypothesis that vagal C-fibres comprise at least two distinct phenotypes. Histological and extracellular electrophysiological experiments revealed that vagal C-fibres innervating the pulmonary system are derived from cell bodies situated in two distinct vagal sensory ganglia. The jugular (superior) ganglion neurones project C-fibres to both the extrapulmonary airways (larynx, trachea and bronchus) and the lung parenchymal tissue. By contrast, C-fibres from nodose (inferior) neurones innervate primarily structures within the lungs. Histologically, nodose neurones projecting lung C-fibres were different from the jugular neurones in that they were significantly less likely to express neurokinins. The nerve terminals within the lungs of both nodose and jugular C-fibres responded with action potential discharge to capsaicin and bradykinin application, but only the nodose C-fibre population responded with action potential discharge to the P2X selective receptor agonist alpha,beta-methylene-ATP. Whole cell patch clamp recording of capsaicin-sensitive nodose and jugular ganglion neurones retrogradely labelled from the lung tissue revealed that, like the nerve terminals, lung specific nodose C-fibre neurones express functional P2X receptors, whereas lung specific jugular C-fibres do not. The data support the hypothesis that both neural crest-derived neurones (jugular ganglia) and placode-derived neurones (nodose ganglia) project C-fibres in the vagus, and that these two C-fibre populations represent distinct phenotypes.

The integrative membrane properties of human bronchial parasympathetic Ganglia neurons.

Parasympathetic ganglia neurons in the lower airway of laboratory animals have membrane properties associated with integration of signals from the central nervous system. In this study, intracellular recordings were made from parasympathetic ganglia located on bronchi from human lungs in order to determine the level of integration provided by human neurons. Ganglion neurons were characterized as either tonic or phasic: tonic neurons responded with repetitive action potentials sustained throughout a depolarizing current step whereas phasic neurons generated one action potential and accommodated. Phasic neurons could be further differentiated as having either short or long duration after hyperpolarizing potentials following single action potentials. In phasic neurons, stimulation of preganglionic nerves elicited one or two populations of nicotinic fast excitatory postsynaptic potentials (fEPSPs) that were graded in amplitude, subthreshold for action potential generation, and decreased in amplitude during higher frequency stimulation. In tonic neurons, single preganglionic stimuli evoked two to five populations of fEPSPs, one to three of which were at threshold for action potential generation. Dye injection into the neurons revealed multiple, branching dendrites. These results provide evidence that human bronchial ganglion neurons have unique membrane properties and anatomical characteristics associated with integrating presynaptic stimuli. Changes in these properties may thus affect output from these ganglia and, consequently, autonomic tone in the lower airways.

Cutting edge: altered pulmonary eosinophilic inflammation in mice deficient for Clara cell secretory 10-kDa protein.

Clara cell secretory protein (CC10) is a steroid-inducible protein, and its in vivo function is currently unclear. The role of CC10 in modulation of pulmonary allergic inflammation was examined in mice deficient for the CC10 gene. Wild-type and homozygous CC10-deficient mice were sensitized with an Ag, OVA, and challenged with either OVA or saline. When compared with that seen in wild-type mice, a significantly higher level of pulmonary eosinophilia was found in Ag-sensitized and challenged CC10-deficient mice. Significantly increased levels of Th2 cytokines IL-4, IL-5, IL-9, and IL-13 were also found in CC10-deficient mice. In addition, an increased level of eotaxin, but not RANTES, was also seen in CC10-deficient mice. No significant difference was observed in the level of a Th1 cytokine, IFN-gamma, between different groups of mice. These results provided the first in vivo evidence that CC10 plays a role in the modulation of pulmonary allergic inflammation.

Transmission in autonomic ganglia.

The activity of airway smooth muscle, glands and vasculature is under tonic control by the autonomic nervous system. Information regarding the function and state of the airway (e.g. blood flow, temperature, oxygen levels, movement, irritants, inflammation, etc.) is relayed to the central nervous system (CNS) in the form of action potentials carried by sensory nerves. This input is integrated at many levels in the CNS and this information is ultimately transformed into coded action potentials carried by various preganglionic nerve pathways from the CNS to peripheral clusters of neurons referred to as autonomic ganglia. In the autonomic ganglia the CNS-derived action potentials cause the release of neurotransmitter(s) at a synapse between the preganglionic nerve terminal and the principal ganglion neuron. The fact that synaptic transmission exists makes the ganglion neuron the final site of integration in this complex reflex pathway. Whether this transmission of information from the CNS occurs, by activating the autonomic ganglion neuron and consequently the effector organ, depends on neurochemical, anatomical, and electrophysiological factors within the ganglion that is the subject of this review.

Neural integration and allergic disease.

Changes in neural activity play a key role in many symptoms of allergic disease, including sneezing, coughing, itching, and ocular irritation, among others. The mechanisms underlying allergen-induced changes in neural activity (reflexes) are largely unknown and under active investigation. Allergic inflammation can affect neural activity on a variety of levels, including at the primary afferent sensory nerve, integrative centers of the central nervous system, autonomic ganglia, and autonomic neuroeffector junction. At the level of the afferent sensory nerve, mediators released after allergen exposure either directly or indirectly increase neuronal firing. At the level of sensory ganglia, which contain cell bodies that innervate a variety of organs, changes in neuronal excitability may lead to a generalization of allergic symptoms. In the central nervous system, where afferent inputs from throughout the body converge, allergic inflammation may be associated with central sensitization, leading to the modulation of the neural reflexes. Finally, at the autonomic ganglia and neuroeffector junction, allergic inflammation appears to be associated with enhanced ganglionic transmission and neurotransmitter release, respectively. Mechanisms by which allergen challenge affects neuronal activity at various levels of the nervous system are reviewed, with a primary emphasis on studies of airway physiologic factors.

Nerve growth factor-induced phenotypic switch in guinea pig airway sensory neurons.

Immunohistochemistry was combined with retrograde tracing techniques to characterize the effect of nerve growth factor (NGF) on substance P (SP) producing vagal neurons innervating the guinea pig trachea. Fast blue dye instilled into the trachea retrogradely labeled nerve cell bodies located in the nodose and jugular ganglia. In untreated guinea pigs > 99% of the SP-containing neurons labeled with fast blue were located in the jugular ganglia. The SP-positive neurons were small in diameter (23 +/- 1 microm) and were negative for neurofilament immunoreactivity. The fast-blue-positive neurons in the nodose ganglia, by contrast, were large in diameter (40 +/- 3 microm) and were negative for SP immunoreactivity and positive for neurofilament immunoreactivity. After NGF-beta injections into the tracheal wall, approximately 10% of the large-diameter nodose neurofilament-positive neurons projecting fibers to the trachea became SP-positive (p < 0.05). We previously demonstrated that nodose nerve endings supplying the trachea are exquisitely mechanically sensitive, but capsaicin- and bradykinin-insensitive. These results suggest that NGF not only increases SP expression in airway neurons, but changes the neuronal phenotype such that large, capsaicin-insensitive nodose neurons with fast-conducting "Adelta" fibers provide a component of the tachykinergic innervation.

Anatomical characteristics of tonic and phasic postganglionic neurons in guinea pig bronchial parasympathetic ganglia.

Anatomical characteristics of principal parasympathetic ganglia neurons on the guinea pig primary bronchus were analyzed, and the procedure for localizing the ganglia without the aid of staining for in vitro physiological studies is described. The neurons were tightly packed within a perineural sheath, and the cell bodies formed a homogeneous population based on size and shape. By using intracellular electrophysiological recordings, unstained neurons within these ganglia were characterized with suprathreshold depolarizing stimuli as having either accommodating action potential patterns (phasic neurons) or repetitive action potential patterns (tonic neurons). After determining whether a cell was tonic or phasic, it was injected with either horseradish peroxidase or Neurobiotin for characterization of its dendrites. There were no differences between tonic and phasic neurons, and both exhibited the following: (1) dendrites were multiple and branching; (2) all processes (axon and dendrites) arose from a circumscribed area on the somatic surface; (3) the initial direction of the processes was usually toward the center of the ganglion, creating a very dense intraganglionic neuropil; (4) tapering processes (presumed dendrites) extended beyond the border of the perineural sheath; and (5) many processes terminated with bouton-like swellings near the somatic surfaces of neighboring neurons within the same ganglion. Electron microscopic examination of dendritic and cell body membranes revealed that greater than 90% of the synapses occurred on dendrites. Based on immunohistochemical staining, all neurons were calbindin negative. These results indicate a relatively homogeneous population of neurons in bronchial parasympathetic ganglia displaying dendritic characteristics compatible with complex integrative properties.

Effect of bradykinin on membrane properties of guinea pig bronchial parasympathetic ganglion neurons.

The effect of bradykinin on membrane properties of parasympathetic ganglion neurons in isolated guinea pig bronchial tissue was studied using intracellular recording techniques. Bradykinin (1-100 nM) caused a reversible membrane potential depolarization of ganglion neurons that was not associated with a change in input resistance. The selective bradykinin B(2) receptor antagonist HOE-140 inhibited bradykinin-induced membrane depolarizations. Furthermore, the cyclooxygenase inhibitor indomethacin attenuated bradykinin-induced membrane depolarizations to a similar magnitude ( approximately 70%) as HOE-140. However, neurokinin-1 and -3 receptor antagonists did not have similar inhibitory effects. The ability of bradykinin to directly alter active properties of parasympathetic ganglion neurons was also examined. Bradykinin (100 nM) significantly reduced the duration of the afterhyperpolarization (AHP) that followed four consecutive action potentials. The inhibitory effect of bradykinin on the AHP response was reversed by HOE-140 but not by indomethacin. These results indicate that bradykinin can stimulate airway parasympathetic ganglion neurons independent of sensory nerve activation and provide an alternative mechanism for regulating airway parasympathetic tone.

Adaptation of guinea-pig vagal airway afferent neurones to mechanical stimulation.

1. Intracellular and extracellular electrophysiological recording techniques were employed to examine the mechanisms involved in adaptation of guinea-pig airway sensory neurones to suprathreshold mechanical stimulation in vitro. Extracellular recordings performed using an in vitro airway preparation revealed two unambiguously distinct subsets of mechanically sensitive nerve endings in the trachea/bronchus. In one group of fibres, the mechanical stimulus caused a brief burst of action potentials, after which the nerve rapidly adapted. In the other group of fibres, repetitive action potentials were evoked as long as the stimulus was maintained above threshold. 2. The adaptation response strictly correlated with ganglionic origin of the soma. Those fibres derived from the nodose ganglion adapted rapidly, whereas those derived from the jugular ganglion were slowly or non-adapting. 3. Intracellular recordings from airway-identified neurones in isolated intact ganglia revealed that the majority of neurones within either the nodose or jugular ganglion adapted rapidly to prolonged suprathreshold depolarizing current injections. 4. The electrophysiological adaptation of nodose ganglion-derived neurones following prolonged suprathreshold current steps was greatly reduced by 4-aminopyridine. However, 4-aminopyridine did not affect the adaptation of rapidly adapting nodose ganglion-derived nerve endings in response to mechanical stimuli. 5. The data suggest that ganglionic origin dictates adaptive characteristics of guinea-pig tracheal and mainstem bronchial afferent neurones in response to mechanical stimulation. Also, the rapid adaptation of nodose nerve endings in the trachea observed during a mechanical stimulus does not appear to be related to the adaptation observed at the soma during prolonged suprathreshold depolarizing current injections.

Characterization of vagal afferent subtypes stimulated by bradykinin in guinea pig trachea.

In vitro electrophysiological techniques were used to examine the effect of bradykinin on guinea pig trachea and bronchus afferent nerve endings arising from the nodose or jugular ganglia. The data reveal that bradykinin activates nerve terminals of jugular C and Adelta fibers. Although the fibers were too few in number to study rigorously, bradykinin also stimulated nodose C fibers innervating the trachea and bronchus. In contrast, Adelta fibers arising from the nodose ganglion were unresponsive to bradykinin challenge. The responses in both jugular C and Adelta fiber types were blocked by a selective bradykinin B2 receptor antagonist and were not dependent on the efferent release of sensory neuropeptides. These data indicate that the sensitivity of guinea pig airway afferent fibers to bradykinin is dependent more on the ganglionic origin of the cell body than on the conduction velocity of its axon.

Ca2+ and K+ currents regulate accommodation and firing frequency in guinea pig bronchial ganglion neurons.

Intracellular microelectrode recordings were obtained from neurons located in adult guinea pig bronchial parasympathetic ganglia in situ to determine the calcium and potassium currents regulating repetitive action potential activity and firing rates by these neurons. Neurons in these ganglia respond to prolonged suprathreshold depolarizing current steps with either a burst of action potentials at the onset of the stimulus (accommodating or phasic neurons) or repetitive action potentials throughout the stimulus (nonaccommodating or tonic neurons). Instantaneous and adapted firing rates during prolonged threshold and suprathreshold stimuli were lower in tonic than in phasic neurons, indicating a longer interspike interval between repetitive action potentials in tonic neurons. In tonic neurons, blockade of A-type current with 4-aminopyridine increased accommodation; 4-aminopyridine or apamin decreased the interspike interval in tonic neurons. Calcium-free buffer, cadmium ions, or omega-conotoxin GVIA also increased accommodation in tonic neurons but did not affect the interspike interval; nifedipine or verapamil did not affect the tonic firing pattern. Accommodation in phasic neurons could be decreased by a conditioning hyperpolarization step of the resting potential, which could be subsequently blocked by 4-aminopyridine or calcium-free buffer. Accommodation in phasic neurons could also be decreased by apamin or barium ions: the repetitive action potentials observed during these treatments could be reversed by cadmium ions or calcium-free buffer. These results indicate that tonic and phasic neurons in guinea pig bronchial parasympathetic ganglia have similar types of calcium currents, but potassium channels may ultimately regulate the accommodation pattern, the firing rate, and, consequently, the output from these neurons.

Inhibition of 5-lipoxygenase diminishes neurally evoked tachykinergic contraction of guinea pig isolated airway.

The role of endogenous 5-lipoxygenase products in modulating tachykinergic neurotransmission in guinea pig isolated trachea was investigated. Tachykinin-containing afferent nerve fibers were stimulated with either electrical field stimulation or antidromic stimulation of the right vagus nerve. This resulted in contractions of the isolated caudal trachea and bronchus that could be blocked with either tetrodotoxin or a combination of neurokinin-1 and neurokinin-2 receptor antagonists. The 5-lipoxygenase inhibitor ZD 2138 (1 microM) significantly inhibited these neurally mediated tachykinergic contractions, by approximately 50%, yet had no effect on the contractions evoked by stimulating tachykinergic fibers in an action potential-independent fashion with capsaicin or by exogenously applied neurokinin A. The effect of ZD 2138 on action potential-driven tachykinergic contractions was mimicked by pobilukast, pranlukast, montelukast and zafirlukast, four structurally unrelated antagonists of the cysteinyl leukotriene 1 receptor subtype. Pobilukast had no effect on the tachykinergic contraction in tissues pretreated with ZD 2138. Likewise, ZD 2138 had no effect on the tachykinergic contractions in tissues pretreated with pobilukast. Intracellular electrophysiological recording of the membrane properties of jugular ganglion neurons, the source of tachykinins in the guinea pig trachea/bronchus, demonstrated that leukotriene D4 caused a membrane depolarization of vagal afferent C-fiber neurons and an increase in input impedance, both of which were abolished by zafirlukast. Taken together, these data indicate that in the resting guinea pig isolated trachea/bronchus, endogenous 5-lipoxygenase activity leads to the production of cysteinyl leukotrienes that amplify action potential-dependent release of tachykinins from airway afferent nerve fibers.

Effect of nedocromil sodium on neurogenic mechanisms in vitro.

We have developed a guinea pig model that allows monitoring of single afferent nerve fiber activity after stimulation of tracheal nerve endings with various stimuli. Action potentials from nodose or jugular neurons are recorded extracellularly. Previous experiments have shown that the excitability of the nerve endings to, for example, mechanical stimuli can be increased by antigen challenge of presensitized guinea pigs. Nedocromil sodium 10(-4) mol/L significantly reduced by more than 50% the generation of action potentials in C fibers in response to stimulation by capsaicin. The precise mechanism is not known, although the membrane depolarization induced by capsaicin was reduced in the presence of nedocromil sodium. This agent did not have a generalized inhibitory effect on afferent excitability because it had no effect on sensitivity to electrical or mechanical stimuli. Nedocromil sodium did not affect the generation of action potentials in A delta and C fibers in the presence of hypertonic saline solution.

Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways.

1. The present study addressed the hypothesis that jugular and nodose vagal ganglia contain the somata of functionally and anatomically distinct airway afferent fibres. 2. Anatomical investigations were performed by injecting guinea-pig airways with the neuronal tracer Fast Blue. The animals were killed 7 days later, and the ganglia were removed and immunostained with antisera against substance P (SP) and neurofilament protein (NF). In the nodose ganglion, NF-immunoreactive neurones accounted for about 98% of the Fast Blue-labelled cells while in the jugular ganglion they accounted for approximately 48%. SP and NF immunoreactivity was never (n = 100) observed in the same cell suggesting that the antisera labelled distinct populations. 3. Electrophysiological investigations were performed using an in vitro guinea-pig tracheal and bronchial preparation with intact afferent vagal pathways, including nodose and jugular ganglia. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in either ganglion. 4. The nodose ganglion contained the somata of mainly fast-conducting tracheal A delta fibres whereas the jugular ganglion contained equal numbers of C fibre and A delta fibre tracheal afferent somata. The nodose A delta neurones adapted rapidly to mechanical stimulation, had relatively low mechanical thresholds, were not activated by capsaicin and adapted rapidly to a hyperosmotic stimulus. By contrast, jugular A delta and C fibres adapted slowly to mechanical stimulation, were often activated by capsaicin, had higher mechanical thresholds and displayed a slow adaptation to a hyperosmotic stimulus. 5. The anatomical, physiological and pharmacological data provide evidence to support the contention that the vagal ganglionic source of the fibre supplying the airways ultimately dictates its neurochemical and physiological phenotype.

Muscarinic receptor regulation of synaptic transmission in airway parasympathetic ganglia.

Muscarinic receptor regulation of synaptic transmission in guinea pig bronchial parasympathetic ganglia was evaluated with the use of intracellular recording of the intrinsic ganglion neurons. Methacholine (1 microM) decreased the amplitude of vagus nerve-stimulated fast excitatory postsynaptic potentials (fEPSP) by 33 and 46% (at 0.8 and 8.0 Hz, respectively) but had no effect on the amplitude of the depolarizations evoked by a bath-applied nicotinic receptor agonist. Methoctramine (1 microM) inhibited methacholine's effect on fEPSP but alone did not influence the magnitude of the fEPSP evoked by vagus nerve stimulation. Methacholine (10 microM) depolarized a subpopulation of neurons (approximately 4 mV), which was blocked by pirenzepine (0.1 microM). In other neurons, either no effect or a small (1-5 mV) hyperpolarization was noted. Cholinergic contractions of bronchial smooth muscle elicited by electrical field stimulation were potentiated by methoctramine to the same extent as those evoked by vagus nerve (preganglionic) stimulation. The data indicate that M2 receptor activation can lead to inhibition of presynaptic acetylcholine release and consequently a significant inhibition of synaptic transmission in bronchial parasympathetic ganglia. The physiological role of this neuromodulatory effect appears limited, however, when studied in the in vitro setting.

Immunomodulation of afferent neurons in guinea-pig isolated airway.

1. The trachea, larynx and main bronchi with the right vagus nerve and nodose ganglion were isolated from guinea-pigs passively immunized 24 h previously with serum containing anti-ovalbumin antibody. 2. The airways were placed in one compartment of a Perspex chamber for recording of isometric tension while the nodose ganglion and attached vagus nerve were pulled into another compartment. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in the ganglion. Mechanosensitivity of the nerve endings was quantified using calibrated von Frey filaments immediately before and after exposure to antigen (10 micrograms ml-1 ovalbumin). 3. Ten endings responded to the force exerted by the lowest filament (0.078 mN) and were not further investigated. In airways from thirteen immunized guinea-pigs, the mechanical sensitivity of A delta afferent fibres (conduction velocity = 4.3 +/- 0.6 m s-1) was enhanced 4.1 +/- 0.9-fold following airway exposure to antigen (P < 0.005). Mechanical sensitivities of afferent fibres (conduction velocity = 4.3 +/- 0.6 m s-1) from non-immunized control guinea-pig airways were unaffected by antigen (n = 13). 4. Antigen did not overtly cause action potential generation except in one instance when the receptive field was located over the smooth muscle. This ending also responded to methacholine suggesting that spatial changes in the receptive field, induced by muscle contraction, were responsible for the activation. 5. The mediators responsible for these effects are unknown, although histamine, prostaglandins, leukotrienes and tachykinins do not appear to be essential. The increase in mechanical responsiveness was not associated with the smooth muscle contraction since leukotriene C4, histamine and tachykinins, which all caused a similar contraction to antigen, did not affect mechanical thresholds. Moreover, the antigen-induced increases in excitability persisted beyond the duration of the smooth muscle contraction. 6. These results demonstrate that antigen-antibody-mediated inflammatory processes may enhance the excitability of vagal afferent nerve terminals projecting from the airway and thus may contribute to the pathophysiology of allergic airway diseases.