Airway Vagal Preganglionic Neurons Research Articles

Moxonidine inhibits excitatory inputs to airway vagal preganglionic neurons via activation of both α2-adrenoceptors and imidazoline I1 receptors

As an imidazoline I1 receptor agonist with very weak binding affinity for α2-adrenoceptors, moxonidine is commonly used in the treatment of hypertension. Moxonidine also has been implicated to act centrally to reduce airway vagal outflow. However, it is unknown at which central sites moxonidine acts to affect airway vagal activity, and how moxonidine takes effect at synaptic and receptor levels. In this study, airway vagal preganglionic neurons (AVPNs) were retrogradely labeled in neonatal rats from the intrathoracic trachea; retrogradely labeled AVPNs in the external formation of the nucleus ambiguus (NA) were identified in rhythmically active medullary slices using whole-cell patch-clamp techniques; and the effects of moxonidine on the spontaneous excitatory postsynaptic currents (EPSCs) of AVPNs were observed at synaptic level. The results show that moxonidine (10 μmol·L−1) significantly inhibited the frequency of spontaneous EPSCs in both inspiratory-activated and inspiratory-inhibited AVPNs. This effect was partially blocked by SKF-86466 (10 μmol·L−1), a highly selective antagonist of α2-adrenoceptors, or AGN-192403, a selective antagonist of imidazoline I1 receptors, and was completely blocked by efaroxan (10 μmol·L−1), an antagonist of both α2-adrenoceptors and imidazoline I1 receptors. These results demonstrate that moxonidine inhibits the excitatory inputs to AVPNs via activation of both α2-adrenoceptors and imidazoline I1 receptors, and suggest that physiologically both of these two types of receptors are involved in the central regulation of airway vagal activity at preganglionic level. Moxonidine might be potentially useful in diseases with aberrant airway vagal activity such as asthma and chronic obstructive diseases.

Orexin-A Excites Airway Vagal Preganglionic Neurons via Activation of Orexin Receptor Type 1 and Type 2 in Rats.

Airway vagal nerves play a predominant role in the neural control of the airway, and augmented airway vagal activity is known to play important roles in the pathogenesis of some chronic inflammatory airway diseases. Several lines of evidence indicate that dysfunctional central orexinergic system is closely related to the severity of airway diseases, however, whether orexins affect airway vagal activity is unknown. This study investigates whether and how orexin-A regulates the activity of medullary airway vagal preganglionic neurons (AVPNs). The expression of orexin receptor type 1 (OX1R) and type 2 (OX2R) was examined using immunofluorescent staining. The effects of orexin-A on functionally identified inspiratory-activated AVPNs (IA-AVPNs), which are critical in the control of airway smooth muscle, were examined using patch-clamp in medullary slices of neonatal rats. Airway vagal response to injection of orexin-A into the magna cisterna was examined using plethysmography in juvenile rats. The results show that retrogradely labeled AVPNs were immunoreactive to anti-OX1R antibody and anti-OX2R antibody. Orexin-A dose-dependently depolarized IA-AVPNs and increased their firing rate. In synaptically isolated IA-AVPNs, the depolarization induced by orexin-A was blocked partially by OX1R antagonist SB-334867 or OX2R antagonist TCS OX2 29 alone, and completely by co-application of both antagonists. The orexin-A-induced depolarization was also mostly blocked by Na+/Ca2+ exchanger inhibitor KB-R7943. Orexin-A facilitated the glutamatergic, glycinergic and GABAergic inputs to IA-AVPNs, and the facilitation of each type of input was blocked partially by SB-334867 or TCS OX2 29 alone, and completely by co-application of both antagonists. Injection of orexin-A into the magna cisterna of juvenile rats significantly increased the inspiratory and expiratory resistance of the airway and consequently decreased the dynamic compliance of the lungs, all of which were prevented by atropine sulfate or bilateral vagotomy. These results demonstrate that orexin-A excites IA-AVPNs via activation of both OX1R and OX2R, and suggest that increased central synthesis/release of orexins might participate in the pathogenesis of airway diseases via over-activation of AVPNs.

Asthmatic Augmentation of Airway Vagal Activity Involves Decreased Central Expression and Activity of CD73 in Rats.

The severity of asthma is closely related to the intensity of airway vagal activity; however, it is unclear how airway vagal activity is centrally augmented in asthma. Here we report that in an asthma model of male Sprague-Dawley rats, the expression and activity of ecto-5'-nucleotidase (CD73) were decreased in airway vagal centers, ATP concentration in cerebral spinal fluid was increased, and the inhibitory and excitatory airway vagal responses to intracisternally injected ATP (5 μmol) and CD73 inhibitor AMPCP (5 μmol), respectively, were attenuated. In airway vagal preganglionic neurons (AVPNs) identified in medullary slices of neonatal Sprague-Dawley rats, AMPCP (100 μmol·L-1) caused excitatory effects, as are shown in patch-clamp by depolarization, increased neuronal discharge, and facilitated spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, exogenous ATP (100 μmol·L-1, 1 mmol·L-1) primarily caused inhibitory effects, which are similar to those induced by exogenous adenosine (100 μmol·L-1). Adenosine A1 receptor antagonist CPT (5 μmol·L-1) blocked the inhibition of sEPSCs induced by 100 μmol·L-1 exogenous ATP and that by 100 μmol·L-1 exogenous adenosine, whereas 50 μmol·L-1 CPT converted the inhibition of sEPSCs induced by 1 mmol·L-1 ATP to facilitation that was blocked by addition of P2X receptor antagonist PPADS (20 μmol·L-1). These results demonstrate that in rat, the sEPSCs of AVPNs are facilitated by extracellular ATP via activation of P2X receptors and inhibited by extracellular adenosine via activation of A1 receptors; in experimental asthma, decreased CD73 expression and activity in airway vagal centers contribute to the augmentation of airway vagal activity through imbalanced ATP/ADO modulation of AVPNs.

Central inspiratory activity rhythmically activates synaptic currents of airway vagal preganglionic neurons in neonatal rats

The airway vagal preganglionic neurons (AVPNs) in the external formation of the nucleus ambiguus (eNA) can be separated into inspiratory-activated AVPNs (IA-AVPNs) and inspiratory-inhibited AVPNs (II-AVPNs). IA-AVPNs are activated by excitatory presynaptic inputs during inspiratory bursts, but the composition and the roles of these excitatory inputs still remain obscure. II-AVPNs are inhibited by inhibitory presynaptic inputs but whether these inhibitory inputs are regulated by excitatory inputs is also unclear. In the current study, AVPNs were retrogradely fluorescent labeled. The IA-AVPNs were discriminated from II-AVPNs by their different synaptic inputs during inspiratory bursts. The excitatory inputs to IA-AVPNs and the presynaptic regulation of II-AVPNs were examined by whole-cell patch clamping. Topical application of 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the recorded IA-AVPNs almost abolished the tonic EPSCs during inspiratory intervals, inhibited the phasic excitatory currents during inspiratory bursts and attenuated the phasic inspiratory inward currents (PIICs) driven by central inspiratory activity. Blockade of α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) respectively inhibited PIICs in some IA-AVPNs. Carbenoxolone, a gap junction uncoupler, partly inhibited the PIICs of IA-AVPNs. Focal application of CNQX to the II-AVPNs significantly inhibited the frequency, peak amplitude and area of the phasic inspiratory outward currents (PIOCs). These findings demonstrated that glutamatergic non-NMDA receptors played a predominant role in the excitatory drive to the IA-AVPNs, and that α4β2, α7 nAChRs and gap junctions were also rhythmically activated by central inspiratory activity. Additionally, glycinergic neurons making inhibitory inputs to the II-AVPNs were pre-synaptically facilitated by excitatory glutamatergic synaptic inputs.

Inspiratory-Activated Airway Vagal Preganglionic Neurones Excited by Thyrotropin-Releasing Hormone via Multiple Mechanisms in Neonatal Rats.

The airway vagal preganglionic neurons (AVPNs) providing projections to intrinsic tracheobronchial ganglia are considered to be crucial to modulation of airway resistance in physiological and pathological states. AVPNs classified into inspiratory-activated AVPNs (IA-AVPNs) and inspiratory-inhibited AVPNs (II-AVPNs) are regulated by thyrotropin-releasing hormone (TRH)-containing terminals. TRH causes a direct excitatory current and attenuates the phasic inspiratory glycinergic inputs in II-AVPNs, however, whether and how TRH influences IA-AVPNs remains unknown. In current study, TRH regulation of IA-AVPNs and its mechanisms involved were investigated. Using retrogradely fluorescent labeling method and electrophysiology techniques to identify IA-AVPNs in brainstem slices with rhythmic inspiratory hypoglossal bursts recorded by a suction electrode, the modulation of TRH was observed with patch-clamp technique. The findings demonstrate that under voltage clamp configuration, TRH (100 nM) caused a slow excitatory inward current, augmented the excitatory synaptic inputs, progressively suppressed the inhibitory synaptic inputs and elicited a distinctive electrical oscillatory pattern (OP). Such a current and an OP was independent of presynaptic inputs. Carbenoxolone (100 μM), a widely used gap junction inhibitor, fully suppressed the OP with persistence of TRH-induced excitatory slow inward current and augment of the excitatory synaptic inputs. Both tetrodotoxin (1 μM) and riluzole (20 μM) functioned to block the majority of the slow excitatory inward current and prevent the OP, respectively. Under current clamp recording, TRH caused a slowly developing depolarization and continuously progressive oscillatory firing pattern sensitive to TTX. TRH increased the firing frequency in response to injection of a square-wave current. The results suggest that TRH excited IA-AVPNs via the following multiple mechanisms: (1) TRH enhances the excitatory and depresses the inhibitory inputs; (2) TRH induces an excitatory postsynaptic slow inward current; (3) TRH evokes a distinctive OP mediated by gap junction.

Corticotropin-releasing hormone modulates airway vagal preganglionic neurons of Sprague–Dawley rats at multiple synaptic sites via activation of its type 1 receptors: Implications for stress-associated airway vagal excitation

Corticotropin-releasing hormone release is the final common pathway of stress-associated neuroendocrine responses. This study tested how corticotropin-releasing hormone modulates airway vagal preganglionic neurons. Airway vagal preganglionic neurons in neonatal rats were retrogradely labeled with fluorescent dye and identified in medullary slices, and their responses to corticotropin-releasing hormone (200nmolL−1) were examined using whole-cell patch clamp. The results show that under current clamp, corticotropin-releasing hormone (200nmolL−1) depolarized airway vagal preganglionic neurons and significantly increased the rate of their spontaneous firing. Under voltage clamp, corticotropin-releasing hormone caused a tonic inward current and significantly facilitated the spontaneous glutamatergic and GABAergic inputs of these neurons. Corticotropin-releasing hormone had no impact on the spontaneous glycinergic inputs of these neurons. In the preexistence of tetrodotoxin (1μmolL−1), corticotropin-releasing hormone had no impact on the miniature excitatory or inhibitory postsynaptic currents, but still induced a tonic inward current and significantly increased the input resistance. The responses induced by corticotropin-releasing hormone were prevented by Antalarmin hydrochloride (50μmolL−1), an antagonist of type 1 corticotropin-releasing hormone receptors, but insensitive to Astressin 2B (200nmolL−1), an antagonist of type 2 corticotropin-releasing hormone receptors. These results suggest that corticotropin-releasing hormone excites airway vagal preganglionic neurons via activation of its type 1 receptors at multiple sites, which includes a direct postsynaptic excitatory action and presynaptic facilitation of both glutamatergic and GABAergic inputs. In stress, corticotropin-releasing hormone might be able to activate the airway vagal nerves and, consequently, participate in induction or exacerbation of airway disorders.

Arginine Vasopressin Alters Both Spontaneous and Phase-Locked Synaptic Inputs to Airway Vagal Preganglionic Neuron via Activation of V1a Receptor: Insights into Stress-Related Airway Vagal Excitation.

The airway vagal preganglionic neurons (AVPNs) in the external formation of the nucleus ambiguus (eNA) play a major role in the vagal control of tracheobronchial smooth muscle tone and maintenance of airway resistance. The eNA receives vasopressinergic projection from the hypothalamic paraventricular nucleus (PVN), the key node for the genesis of psychological stress. Since airway vagal excitation is reportedly to be associated with the psychological stress-induced/exacerbated airway hyperresponsiveness in asthmatics, arginine vasopressin (AVP) might be involved in stress-related airway vagal excitation. However, this possibility has not been validated. This study aimed to test whether and how AVP regulates AVPNs. In rhythmically active medullary slices of newborn rats, retrogradely labeled AVPNs were identified as inspiratory-activated and inspiratory-inhibited AVPNs (IA- and II-AVPNs) using patch-clamp techniques according to their inspiratory-related firing behavior and synaptic activities. The results show that under current clamp, AVP depolarized both IA- and II-AVPNs, and significantly increased their spontaneous firing rate. Under voltage clamp, AVP elicited a slow inward current, and significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in both types of AVPNs. In addition, AVP significantly enhanced the phase-locked excitatory inspiratory inward current in inspiratory-activated airway vagal preganglionic neurons (IA-AVPNs), but significantly suppressed the phase-locked inhibitory inspiratory outward current in II-AVPNs. In both types AVPNs, AVP significantly increased the frequency and amplitude of pharmacologically isolated spontaneous GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs). All of the AVP-induced effects were prevented by SR49059, an antagonist of V1a receptors, but unaffected by SSR149415, an antagonist of V1b receptors. AVP did not cause significant changes in the miniature excitatory postsynaptic currents (mEPSCs), miniature inhibitory postsynaptic currents (mIPSCs) and membrane input resistance of either type of AVPNs. These results demonstrate that AVP, via activation of V1a receptors, enhanced the spontaneous excitatory and inhibitory inputs similarly in the two types of AVPNs, but differentially altered their phase-locked inspiratory excitatory and inhibitory inputs. The overall effects of AVP are excitatory in both types AVPNs. These results suggest that increased central AVP release may be involved in the stress-induced augmentation of airway vagal activity, and, consequently, the induction or exacerbation of some airway diseases.

Activation of α1-adrenoceptors facilitates excitatory inputs to medullary airway vagal preganglionic neurons.

In mammals, the neural control of airway smooth muscle is dominated by a subset of airway vagal preganglionic neurons in the ventrolateral medulla. These neurons are physiologically modulated by adrenergic/noradrenergic projections, and weakened α₂-adrenergic inhibition of them is indicated to participate in the pathogenesis and exacerbation of asthma. This study tests whether these neurons are modulated by α₁-adrenoceptors, and if so, how. In anesthetized adult rats, microinjection of the α₁A-adrenoceptor agonist A61603 (1 pmol) unilaterally into the medullary region containing these neurons caused a significant increase in airway resistance, which was prevented by intraperitoneal atropine (0.5 mg/kg). In rhythmically firing medullary slices of newborn rats, A61603 (10 nM) caused depolarization in both the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons that were retrogradely labeled, and a significant increase in the spontaneous firing rate. Under voltage clamp, A61603 significantly enhanced the spontaneous excitatory inputs to both types of neurons and caused a tonic inward current in the inspiratory-activated neurons along with significantly increased peak amplitude of the inspiratory inward currents. The responses in vitro were prevented by α₁A-adrenoceptor antagonist RS100329 (1 μM), which alone significantly inhibited the spontaneous excitatory inputs to both types of the neurons. After pretreatment with tetrodotoxin (1 μM), A61603 (10 or 100 nM) had no effect on either type of neuron. We conclude that in rats, activation of α₁-adrenoceptors in the medullary region containing airway vagal preganglionic neurons increases airway vagal tone, and that this effect is primarily mediated by facilitation of the excitatory inputs to the preganglionic neurons.

Nicotine enhances both excitatory and inhibitory synaptic inputs to inspiratory‐activated airway vagal preganglionic neurons

The airway vagal preganglionic neurons (AVPNs) supply the essential excitatory drive to the postganglionic neurons and dominate the neural control of the airway both physiologically and pathophysiologically. The AVPNs express multiple subunits of nicotinic acetylcholine receptors (nAChRs), but the influences of exogenous nicotine and endogenous acetylcholine are unknown. This study examined the effects of nicotine and endogenous acetylcholine on retrogradely labelled, functionally identified inspiratory-activated AVPNs (IA-AVPNs) using the patch-clamp technique. Nicotine (10 μmol l(-1)) significantly increased the frequency and amplitude of the spontaneous EPSCs of IA-AVPNs, and these effects were insensitive to methyllycaconitine (MLA, 100 nmol l(-1)), an antagonist of the α7 type of nAChR, but was prevented by dihydro-β-erythroidine (DHβE, 3 μmol l(-1)), an antagonist of the α4β2 type of nAChR. Nicotine caused a tonic inward current in IA-AVPNs, which was reduced by MLA or DHβE alone, but was not abolished by co-application of MLA and DHβE. Nicotine caused a significant increase in the frequency of GABAergic and glycinergic spontaneous IPSCs and significantly increased the amplitude of glycinergic spontaneous IPSCs, all of which were prevented by DHβE. Nicotine had no effects on the miniature EPSCs or miniature IPSCs following pretreatment with TTX. Under current clamp, nicotine caused depolarization and increased the firing rate of IA-AVPNs during inspiratory intervals. Neostigmine (10 μmol l(-1)), an acetylcholinesterase inhibitor, mimicked the effects of nicotine. These results demonstrate that nicotine and endogenous ACh enhance the excitatory and inhibitory synaptic inputs of IA-AVPNs and cause a postsynaptic excitatory current and that the nicotinic effects are mediated presynaptically by activation of the α4β2 type of nAChR and postsynaptically by activation of multiple nAChRs, including α7 and α4β2 types.

Agonist of 5-HT1A/7 receptors but not that of 5-HT2 receptors disinhibits tracheobronchial-projecting airway vagal preganglionic neurons of rats

The vagus nerves supply the major cholinergic tone to airway smooth muscles physiologically and play critical roles in the genesis of airway hyperreactivity under some pathological conditions. Postganglionic airway cholinergic tone relies largely on the ongoing activity of medullary airway vagal preganglionic neurons (AVPNs), of which the tracheobronchial-projecting ones are primarily located in the external formation of the nucleus ambiguus (eNA). AVPNs are regulated by 5-HT, and 5-HT1A/7 and 5-HT2 receptors have been indicated to be involved. But the mechanisms at synaptic level are unknown. In the present study, tracheobronchial-projecting AVPNs (T-AVPNs) were retrogradely labeled from the trachea wall; fluorescently labeled T-AVPNs in the eNA were recorded with whole-cell voltage patch clamp; and the effects of 5-HT1A/7 receptor agonist (±)-8-Hydroxy-2-(dipropylamino) tetralin hydrobromide (8-OH-DPAT) (1 μmol L−1) and 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10 μmol L−1) on the synaptic inputs were examined. 8-OH-DPAT significantly inhibited the GABAergic and glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) of T-AVPNs in both the frequency and amplitude but had no effect on the GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs). The 8-OH-DPAT inhibition of the GABAergic and glycinergic sIPSCs was prevented by 5-HT1A/7 receptor antagonist N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl] ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY-100635) (1 μmol L−1). 8-OH-DPAT had no effect on the glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) and caused no alterations in the baseline current and input resistance of T-AVPNs. DOI had no effect on any types of the synaptic inputs of T-AVPNs. These results suggest that 5-HT1A/7 receptor agonist causes “disinhibition” of T-AVPNs, which might, in part, account for the reflex increase of airway resistance.

Inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons in the ventrolateral medulla of neonatal rat are different in intrinsic electrophysiological properties

This study investigates the firing properties of the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons located in the external formation of the nucleus ambiguus. The results showed that inspiratory-activated and inspiratory-inhibited neurons are distributed with different density and site preference in this area. Inspiratory-inhibited neurons exhibit significantly more positive resting membrane potential, more negative voltage threshold and lower minimal current required to evoke an action potential under current clamp. The afterhyperpolarization in inspiratory-activated neurons was blocked by apamin, a blocker of the small-conductance Ca(2+)-activated K(+) channels; and that in inspiratory-inhibited neurons by charybdotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) channels. Under voltage clamp, depolarizing voltage steps evoked tetrodotoxin-sensitive rapid inward sodium currents, 4-aminopyridine-sensitive outward potassium transients and lasting outward potassium currents. 4-Aminopyridine partially blocked the lasting outward potassium currents of inspiratory-activated neurons but was ineffective on those of inspiratory-inhibited neurons. These findings suggest that inspiratory-activated and inspiratory-inhibited neurons are differentially organized and express different types of voltage-gated ion channels.

Thyrotropin-releasing hormone causes a tonic excitatory postsynaptic current and inhibits the phasic inspiratory inhibitory inputs in inspiratory-inhibited airway vagal preganglionic neurons

The airway vagal preganglionic neurons (AVPNs) in the external formation of the nucleus ambiguus (eNA), which include the inspiratory-activated AVPNs (IA-AVPNs) and inspiratory-inhibited AVPNs (II-AVPNs), predominate in the control of the trachea and bronchia. The AVPNs receive particularly dense inputs from terminals containing thyrotropin-releasing hormone (TRH). TRH microinjection into the nucleus ambiguus (NA) caused constriction of the tracheal smooth muscles. However, it is unknown whether TRH affects all subtypes of the AVPNs in the eNA, and as a result affects the control of all types of target tissues in the airway (smooth muscles, submucosal glands, and blood vessels). It is also unknown how TRH affects the AVPNs at neuronal and synaptic levels. In this study, the AVPNs in the eNA were retrogradely labeled from the extrathoracic trachea, the II-AVPNs were identified in rhythmically firing brainstem slices, and the effects of TRH were examined using patch-clamp. TRH (100 nmol L−1) enhanced both the rhythm and the intensity of the hypoglossal bursts, and caused a tonic excitatory inward current in the II-AVPNs at a holding voltage of −80 mV. The frequency of the spontaneous excitatory postsynaptic currents (EPSCs) in the II-AVPNs, which showed no respiratory-related change in a respiratory cycle, was not significantly changed by TRH. At a holding voltage of −50 mV, the II-AVPNs showed both spontaneous and phasic inspiratory (outward) inhibitory postsynaptic currents (IPSCs). TRH had no effect on the spontaneous IPSCs but significantly attenuated the phasic inspiratory outward currents, in both the amplitude and area. After focal application of strychnine, an antagonist of glycine receptors, to the II-AVPNs, the spontaneous IPSCs were extremely scarce and the phasic inspiratory inhibitory currents were abolished; and further application of TRH had no effect on these currents. Under current clamp configuration, TRH caused a depolarization and increased the firing rate of the II-AVPNs during inspiratory intervals. These results demonstrate that TRH affects the II-AVPNs both postsynaptically via a direct excitatory current and presynaptically via attenuation of the phasic glycinergic synaptic inputs.