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

Journal Pre-proofs Research report Moxonidine inhibits excitatory inputs to airway vagal preganglionic neurons via activation of both α2-adrenoceptors and imidazoline I1 receptors Xujiao Zhou, Ding He, Xianxia Yan, Xingxin Chen, Rui Li, Guangming Zhang, Jijiang Wang PII: DOI: Reference:

S0006-8993(20)30051-2 https://doi.org/10.1016/j.brainres.2020.146695 BRES 146695

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Brain Research

Received Date: Revised Date: Accepted Date:

9 August 2019 26 December 2019 29 January 2020

Please cite this article as: X. Zhou, D. He, X. Yan, X. Chen, R. Li, G. Zhang, J. Wang, Moxonidine inhibits excitatory inputs to airway vagal preganglionic neurons via activation of both α2-adrenoceptors and imidazoline I1 receptors, Brain Research (2020), doi: https://doi.org/10.1016/j.brainres.2020.146695

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Title: Moxonidine inhibits excitatory inputs to airway vagal preganglionic neurons via activation of both 2-adrenoceptors and imidazoline I1 receptors Running title: Moxonidine modulation of airway vagal neurons Authors: Xujiao Zhou1a-c; Ding He2; Xianxia Yan2, Ph.D; Xingxin Chen2; Rui Li3, Guangming Zhang4* Jijiang Wang, Ph.D2* Author Contributions Jijiang Wang designed the study and prepared the manuscript; Xujiao Zhou, Ding He, Xianxia Yan, Xingxin Chen, and Rui Li performed the experiments, analyzed the data and helped preparation of the manuscript; Guangming Zhang helped interpretation of the data and preparation of the manuscript. Affiliations: 1a, Eye Institute in Eye & ENT Hospital, and NHC Key Laboratory of Myopia, Fudan University. 1b, Shanghai Key Laboratory of Visual Impairment and Restoration. 1c, Key Laboratory of Myopia, Chinese Academy of Medical Sciences. 2. Department of Physiology and Pathophysiology, Fudan University School of Basic Medical Sciences. 3. Department of Nursing, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China. 4. Department of Anesthesiology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China. *Corresponding authors: Jijiang Wang, Ph.D: Department of Physiology and Pathophysiology, Fudan University School of Basic Medical Sciences. 130 Dong’an Rd., 207 seventh building, west campus, Shanghai 200032, China. Tel: 8621-54237405. Guangming Zhang, MD, Department of Anesthesiology, Tongren

Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China.

Tel:

8621-52039999.

Abstract As an imidazoline I1 receptor agonist with very weak binding affinity for 2-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 mol · 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 mol · L−1), a highly selective antagonist of 2-adrenoceptors, or AGN-192403, a selective antagonist of imidazoline I1 receptors, and was completely blocked by efaroxan (10 mol · L−1), an antagonist of both 2-adrenoceptors and imidazoline I1 receptors. These results demonstrate that moxonidine inhibits the excitatory inputs to AVPNs via activation of both 2-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. Key words: moxonidine; airway; vagal; neuron; patch-clamp

Abbreviations ACSF, artificial cerebrospinal fluid AVPN, airway vagal preganglionic neuron COPD, chronic obstructive pulmonary diseases eNA, external formation of the nucleus ambiguus GABA, -aminobutyric acid IA-AVPN, inspiratory-activated airway vagal preganglionic neuron II1-R, imidazoline I1 receptor II-AVPN, inspiratory-inhibited airway vagal preganglionic neuron NA, nucleus ambiguus

1. Introduction Imidazoline I1 receptor (II1-R) agonists, such as moxonidine and rilmenidine, are a class of centrally-acting antihypertension drugs. These drugs have far more potent binding affinity to II1-Rs than to 2-adrenoceptors, and are thought to be more superior in the treatment of hypertension (Schafer et al., 1995; Szabo, 2002). Several lines of evidence have indicated that moxonidine, of which the binding affinity to II1-Rs is 40-70 folds more potent than that to 2-adrenoceptors (Ernsberger et al., 1993), might also act centrally to affect airway vagal outflow (Haxhiu et al., 1998; Haxhiu et al., 1999). However, it is unclear which brain sites are acted, and whether II1-Rs are independently involved. In mammals including humans, the neural regulation of the lower airway is dominated by the pulmonary branch of the vagus nerves (Canning, 2006; van der Velden and Hulsmann, 1999). Airway vagal preganglionic neurons (AVPNs) are the final common pathway through which the brain regulates the tonic and reflexive airway vagal outflow. These neurons originate primarily from three sites in the medulla: the dorsal motor nucleus of the vagus, nucleus ambiguus (NA) and the intermediate zone lying between these two nuclei (Haxhiu et al., 1993; Haxhiu and Loewy, 1996; Jordan, 2001; Kc et al., 2004). However, only those AVPNs located in the external formation of the NA (eNA) have been proved to be able to increase tracheobronchial smooth muscle tone upon experimental activation (Haselton et al., 1992), hence AVPNs in the eNA are thought to be critically important in the control of elastic airway resistance. AVPNs are not intrinsically active, and their spontaneous and phase-locked

inspiratory activities are largely determined by their excitatory inputs. In inspiratory-activated AVPNs (IA-AVPNs), the inspiratory firing is triggered by the phase-locked bursting of excitatory inputs. In both IA-AVPNs and inspiratory-inhibited AVPNs (II-AVPNs), the spontaneous firing during inspiratory intervals is triggered by the spontaneous excitatory inputs (Chen et al., 2007; Chen et al., 2012). II1-Rs are densely expressed in the NA (Ruggiero et al., 1998), indicating that at the level AVPNs, II1-Rs might be independently involved in airway vagal control physiologically. However, this possibility has not been validated experimentally. In addition, asthma and chronic obstructive pulmonary diseases (COPD) are characterized by enhanced airway vagal activity; and patients with combined hypertension and asthma or COPD are far from rare. Elucidation of II1-R-mediated regulation of airway vagal activity is thus essential for the therapeutic use of II1-R agonists in these patients. In the present study, the effects of moxonidine on the excitatory inputs of functionally identified AVPNs are examined using whole-cell patch-clamp techniques. We aimed to test the hypothesis that moxonidine inhibits the excitatory inputs of AVPNs through activation of both 2-adrenoceptors and imidazoline I1 receptors.

2. Results 2.1 Moxonidine significantly inhibited the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in AVPNs Moxonidine (10 mol·L-1) caused a significant decrease in the frequency, but not the amplitude, of spontaneous EPSCs. The moxonidine-induced frequency decrease is by 70% in IA-AVPNs and by 81% in II-AVPNs (Fig.1A1-A4, B1-B4). These responses started within 3 min after moxonidine application and were reversible. Moxonidine did not cause consistent changes in the phasic inspiratory inward current in IA-AVPNs or the phasic inspiratory outward current in II-AVPNs, and did not cause a significant change of the membrane resistance in either IA-AVPNs (75.5 ± 13.2 MΩ before vs. 78.5 ± 13.6 MΩ during moxonidine application; P = 0.35, n = 6; paired Student’s) or II-AVPNs (84.3 ± 7.5 MΩ before vs. 85.6 ± 10.6 MΩ during moxonidine application; P =0.82, n = 5; paired Student’s t-test). Moxonidine typically did not cause a measurable change of the baseline current, except in some neurons an upward shift was observed. It was noticed that in these neurons with a detectable outward change of baseline current, the baseline frequency of spontaneous EPSCs is relatively higher (>5 Hz) and the frequency decrease cause by moxonidine is relatively more conspicuous (Fig.1B1). At the end of the experiments, all of the spontaneous EPSCs were abolished by application of CNQX (50 mol·L-1). 2.2 The moxonidine-induced frequency decrease of spontaneous EPSCs in AVPNs was attenuated by SKF-86466 and AGN-192403, respectively, and abolished by efaroxan

SKF-86466 (10 mol·L-1), a highly selective antagonist of 2-adrenoceptors, caused a significant frequency increase of spontaneous EPSCs alone, in both IA- and II-AVPNs (Fig.2). And, after pretreatment of the slices with SKF-86466, moxonidine (10 mol·L-1) still caused a significant frequency decrease of spontaneous EPSCs, which is by 32% in IA-AVPNs (P < 0.05 compared with that in the absence of SKF-86466, independent t-test) and 48% in II-AVPNs (P < 0.05 compared with that in the absence of SKF-86466, independent t-test). However, compared with the baseline frequency of spontaneous EPSCs, the frequency of spontaneous EPSCs during mixed application of SKF-86466 and moxonidine was not significantly different, in either IA- or IIAVPNs (P > 0.05 for both types of neurons, independent t-test. Fig.2). AGN-192403 (10 mol·L-1), a selective antagonist of II1-Rs, also caused a significant frequency increase of spontaneous EPSCs alone, in both IA- and II-AVPNs (Fig.3). And, after pretreatment of the slices with AGN-192403, moxonidine (10 mol·L-1) still caused a significant frequency decrease of spontaneous EPSCs, which is by 48% in IA-AVPNs (P < 0.05 compared with that in the absence of AGN-192403, independent t-test) and 36% in II-AVPNs (P < 0.05 compared with that in the absence of AGN-192403, independent t-test). Compared with the baseline frequency of spontaneous EPSCs, the frequency of spontaneous EPSCs during mixed application of AGN-192403 and moxonidine was not significantly different, in either IA- or II-AVPNs (P > 0.05 for both types of neurons, independent t-test. Fig.3). In addition, the

moxonidine-induced

frequency

inhibition

of

spontaneous

EPSCs

after

pretreatment with AGN-192403 is not significantly different compared with that after pretreatment with SKF-86466, in either IA- or II-AVPNs (P > 0.05 for both types of neurons, independent t-test). After pretreatment of the slices with efaroxan (10 mol·L-1), moxonidine (10 mol·L-1) no longer caused any significant change in the spontaneous EPSCs of either IA- or II-AVPNs. However, compared with SKF-86466 or AGN-192403, efaroxan alone did not cause any significant change in the spontaneous EPSCs of either IA- or IIAVPNs (Fig.4).

3. Discussion The major finding in the present study is that moxonidine (10 mol·L-1), an agonist of II1-Rs with weak binding affinity to 2-adrenoceptors, caused a significant frequency decrease of spontaneous EPSCs in both IA- and II-AVPNs; and this inhibition was attenuated by 2-adrenoceptor blocker SKF-86466 (10 mol·L-1) and II1-R blocker AGN-192403, respectively, but abolished by efaroxan (10 mol·L-1), an antagonist of mixed 2-adrenoceptors and II1Rs. These results for the first time supply evidence that moxonidine inhibits the excitatory inputs to AVPNs via activation of both 2-adrenoceptors and II1-Rs, and suggest that physiologically both of these two types of receptors are involved in the central regulation of airway vagal activity at preganglionic level. In the moxonidine-induced frequency inhibition of the spontaneous EPSCs in AVPNs, SKF-86466, a highly selective antagonist of 2-adrenoceptors, causes similar attenuation compared with AGN-192403, a selective antagonist of II1-Rs. These results suggest that although the binding affinity of moxonidine to II1-Rs is 40-70 folds more potent than that to 2-adrenoceptors (Ernsberger et al., 1993), activation of 2-adrenoceptors

is

equally

important

with

activation

of

II1-Rs

in

the

moxonidine-induced frequency inhibition of the spontaneous EPSCs in AVPNs. Moxonidine (10 mol·L-1) typically did not cause a measurable change of the baseline current in either IA- or II-AVPNs, except in some neurons of which the baseline frequency of spontaneous EPSCs is relatively higher (>5 Hz) and the frequency decrease cause by moxonidine is relatively more conspicuous. These results suggest

that moxonidine (10 mol·L-1) has little, if any, direct postsynaptic effect on AVPNs, and the outward shift of the baseline current in some of the AVPNs is more likely due to reduced summation of spontaneous EPSCs in response to moxonidine application. This postulation is also supported by the results that moxonidine (10 mol·L-1) did not cause a significant change of the input resistance in either IA- or II-AVPNs. Therefore, in the moxonidine modulation of AVPNs, this study for the first time demonstrates that the action of moxonidine on the presynaptic excitatory inputs plays a critical role. Interestingly, in both IA- or II-AVPNs, SKF-86466 (10 mol·L-1) or AGN-192403 alone caused a significant frequency increase of baseline spontaneous EPSCs, suggesting that the spontaneous EPSCs of AVPNs normally are under tonic inhibition from activation of both 2-adrenoceptors and II1-Rs. However, efaroxan, which blocks both 2-adrenoceptors and II1-Rs, did not cause any significant change of baseline spontaneous EPSCs alone, in either IA- or II-AVPNs. The mechanism involved in these differences is unknown, thus needs further investigation. Asthma and COPD usually show exaggerated airway vagal activity in addition to airway

inflammation.

Agonists

of

II1-Rs

with

varied

binding

affinity

to

2-adrenoceptors, as commonly used drugs for treatment of hypertension, their potential therapeutic use in asthma and COPD has also been investigated previously. However, whether the effects are beneficial or harmful is still in controversy. For example, intravenous (but not inhaled) dexmedetomidine completely blocks the bronchoconstriction induced by histamine in anesthetized dogs (Groeben et al., 2004). When inhaled, clonidine or rilmenidine reduces the immediate bronchial

response to allergens, whereas they aggravate the bronchial response to histamine when ingested (Dinh et al., 1988; Dinh and Lockhart, 1989). Up to date, no study has been performed regarding the potential therapeutic use of moxonidine in asthma and COPD. The present study demonstrates that moxonidine is able to inhibit the excitatory inputs, indicating that moxonidine might be potentially useful in treating asthma or COPD, via inhibition of the increase of airway vagal activity. Since patients with combined hypertension and asthma or COPD are far from rare, it is essential to further investigate the effects and mechanisms of II1-R/2-adrenoceptor agonists including moxonidine in the treatment of asthma and COPD of humans. In rats elder than 10 days, it is now technically hard to keep a medullary slice well oxygenated and rhythmically active in vitro. For this reason, newborn rather than elder rats were used in this study. Therefore, it must be acknowledged that the results of this study may not completely apply to adult rats or other animals. In summary, the present study demonstrates that moxonidine inhibits the excitatory inputs to AVPNs via activation of both 2-adrenoceptors and II1-Rs, 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 COPD.

4. Experimental procedure 4.1 Animals and ethical approval The animal procedures in this study are in accordance with the recommendations of the guidelines for the Care and Use of Laboratory Animals (1996. National Academy of Sciences. Washington, DC, USA), and were approved by the Ethical Committee of the Fudan University School of Basic Medical Sciences (No. 20110307-060 and No. 20170223-073). Newborn Sprague–Dawley (SD) rats together with the feeding mothers were purchased from the Experimental Animal Center of the Chinese Academy of Science in Shanghai, and housed in a temperature-controlled room (22 25°C) illuminated from 07:00 to 19:00. Food and water were available ad libitum. One cage was shared by a single mother rat and her pups. A total of 68 newborn rats of either gender were sacrificed in this study. Maximal efforts were made to minimize the number of animals and their suffering. 4.2 Retrograde fluorescent labeling of AVPNs, preparation of medullary slices and electrophysiological recording AVPNs in 2- to 3-day-old rats were retrogradely labeled from the thoracic segment of the trachea with rhodamine (X-rhodamine-5-(and-6)-isothiocyanate, Molecular Probe) through a surgery under anesthesia of halothane. Twenty-four to 48 h after the surgery, medullary slices (550 m to 750 m thickness) with rhythmic inspiratory-like hypoglossal bursting were cut in cold (4 ℃) artificial cerebrospinal fluid (ACSF) using a vibratome (VT 1000S; Leica Microsystems, Wetzlar, Germany). The composition of the ACSF is as the following (in mmol·L-1): NaCl, 124; KCl, 3.0; KH2PO4, 1.2; CaCl2,

2.4; MgSO4, 1.3; NaHCO3, 26; and D-glucose 10. The ACSF was constantly bubbled with 95% O2 and 5% CO2, and the pH was maintained at 7.4. Slices were transferred to a recording chamber (0.6 ml volume) that was perfused with flowing (6-9 mL·min-1) ACSF. The inspiratory-like activity of the hypoglossal rootlets was recorded using a suction electrode, amplified with a BMA-931 bioamplifier (CWE Inc., Ardmore, PA, USA) (5 kHz sampling frequency; 10-1000 Hz band pass; 20,000 times), and electronically integrated ( = 200 ms) with a MA-1000 Moving Averager (CWE Inc.). The potassium concentration in the flowing ACSF was increased to 8 mmol·L-1 in order to obtain stable rhythm of the inspiratory-like hypoglossal bursts. Bath temperature was maintained at 26 ± 0.5℃. Fluorescently labeled AVPNs in the eNA were identified from the rostral cutting plane of the slices using an Olympus upright microscope [BX51WI, Olympus (China) Co. Ltd., Shanghai, China] with a 40×-water immersion objective lens. AVPNs were recorded under whole-cell patch-clamp configuration. The composition of the pipette solution is as the following (in mmol·L-1): K+ gluconate, 150; HEPES, 10; K+ ATP, 2; MgSO4-7H2O, 2; EGTA, 1; CaCl2, 0.1; Na+ GTP, 0.1, and the pH was 7.3. The pipette resistance and capacitance were not compensated either before or after gaining intracellular access. IA-AVPNs were distinguished by their bursting excitatory inputs, and were normally clamped at -80 mV; and II-AVPNs were distinguished by their bursting inhibitory inputs, and were normally clamped at -50 mV, as has been described in detail previously (Chen et al., 2019). In some experiments, for calculation of input resistance, a 5-mV, 200-ms hyperpolarizing pulse was applied to the AVPN

under recording at a 10-s interval, and the AVPN was recorded discontinuously in sweeps with a sweep interval of 2 s. An Axonpatch 700B amplifier (Molecular Devices LLC, Sunnyvale, CA, USA) (10 kHz sampling frequency; 3 kHz filter frequency) was used to amplify the patch-clamp signal. The patch-clamp signal and integrated hypoglossal activity were collected using the Clampex 10.2 software (Molecular Devices LLC) after digitization with a 1440A digidata (Molecular Devices LLC). All of the procedures used in this section have been described more detailed in our previous studies (Chen et al., 2007; Chen et al., 2012). 4.3 Drug application Drugs

were

used

globally

in

the

bath.

Moxonidine

[4-Chloro-6-methoxy-2-methyl-5-(2-imidazolin-2-yl)aminopyrimidine

hydrochloride hydrochloride]

(10 mol · L−1) was normally applied for 10 min. Application of SKF-86466 hydrochloride

(6-Chloro-N-methyl-2,3,4,5-tetrahydro-1-H-3-benzazepine

hydrochloride) (10 mol L−1), a potent and highly selective antagonist of 2-adrenoceptors,

AGN-192403

hydrochloride

((±)-2-endo-Amino-3-exo-isopropylbicyclo[2.2.1]heptane hydrochloride) (10 mol·L−1), a

selective

antagonist

of

II1-Rs,

or

efaroxan

hydrochloride

[2-Ethyl-2-(imidazolin-2-yl)-2,3-dihydrobenzofuran hydrochloride] (10 mol · L−1), an antagonist of mixed 2- adrenoceptors and II1-Rs, was started 10 min prior to, and throughout

the

subsequent

application

period

of

moxonidine.

CNQX

(6-cyano-7-nitroquinoxaline-2,3-dione) (50 mol·L−1) was applied to block non-NMDA

glutamatergic synaptic currents. Each drug was applied only once in individual slices to avoid desensitization. Thus, only one AVPN was examined in each individual slices. SKF-86466 was purchased from Tocris Bioscience (Bristol, UK). All of the other drugs were purchased from Sigma-Aldrich (St Louis, MO, USA). 4.3 Experimental design and statistical analyses In medullary slices of neonatal rats, functionally identified IA- and II-AVPNs were recorded under voltage clamp to obtain the spontaneous EPSCs and phase-locked inspiratory synaptic currents. The responses of AVPNs to moxonidine were obtained by application of moxonidine to the slices, either alone or after pretreatment with a SKF-86466 or efaroxan. Spontaneous synaptic currents were analyzed with MiniAnalysis (version 4.3.1; Synaptosoft Inc., Fort Lee, NJ, USA) with minimal acceptable amplitude of 10 pA. In each AVPN, the data from a 30-s recording segment during different period of the experiments was analyzed and averaged. During application of moxonidine, either alone or in combination with SKF-86466 or efaroxan, the data during the maximal response period were analyzed. During the period that only SKF-86466 or efaroxan was applied, the data analyzed were selected from a period during which the changes induced by SKF-86466 or efaroxan had been stable. When analyzing the spontaneous EPSCs during inspiratory intervals, the bursting EPSCs during the inspiratory phase were ignored. The phase-locked inspiratory currents and the moxonidine-induced input resistance change of AVPNs were analyzed with the Clampfit 10.2 software (Molecular Devices LLC). Before the phasic inspiratory

currents were analyzed, selected recording segments of the membrane current signal were low-pass filtered at 5 Hz with the eight-pole Bessel filter. At least five consecutive inspiratory phases during control recording and during drug application were averaged for comparison. The input resistance from a period of 50 s (5 traces) during control recording and during the maximal response period was averaged. Data are presented as means  SE. When only two groups of data were compared, paired or independent t-tests was used when appropriate. When more than two groups of data were compared, one-way ANOVA followed by Bonferroni correction was used. Paired t-tests was carried out using the Origin 8.0 software (OriginLab, Northampton, MA); and one-way ANOVA followed by Bonferroni correction was carried out using the SPSS 17.0 software (SPSS, Inc., IL, USA). Significance was set at p < 0.05.

Acknowledgements This study was sponsored by the NSFC (National Natural Science Foundation of China) grant 81970002, 81770003 and 81270060 to J Wang, 81400396 to X Zhou, the Shanghai Natural Science Foundation grant 19ZR1408400 to X Zhou, and the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences 2018PT32019 to X Zhou. Conflict of Interest: The authors declare no competing financial interests. Funding Sources: This study was sponsored by the NSFC (National Natural Science Foundation of China) grant 81970002, 81770003 and 81270060 to J Wang, 81400396 to X Zhou, the Shanghai Natural Science Foundation grant 19ZR1408400 to X Zhou, and the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences 2018PT32019 to X Zhou. .

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Figure legends Figure 1. Moxonidine significantly inhibited the frequency of spontaneous EPSCs in AVPNs. (A1 and B1) Recoding of representative IA-AVPN (A1) and a representative II-AVPN (B1) (upper panels), along with integrated activity of hypoglossal activity (XII, lower panels). (A2 and B2) Recording segments in (A1) and (B1) are shown in an enlarged scale, showing the changes of spontaneous EPSCs induced by moxonidine (10 mol·L-1). (A3, A4 and B3, B4) Summarized data for the frequency (A3, B3) and amplitude (A4, B4) of spontaneous EPSCs in IA-AVPN (A3, A4) and II-AVPNs (B3, B4). Mox., moxonidine. *, P < 0.05; **, P < 0.01. Figure 2. The moxonidine-induced frequency decrease of spontaneous EPSCs in AVPNs was attenuated by SKF-86466. (A1) Recoding of representative IA-AVPN (upper panel), along with integrated activity of hypoglossal activity (XII, lower panel). (A2) The recording segments in (A1) are shown in an enlarged scale, showing that after pretreatment with SKF-86466 (10 mol·L-1), moxonidine (10 mol·L-1) still induced a frequency decrease of spontaneous EPSCs in IA-AVPNs. (B1) Recording segments in a representative II-AVPN, showing that after pretreatment with SKF-86466 (10 mol·L-1), moxonidine (10 mol·L-1) induced a similar frequency decrease of spontaneous EPSCs as in IA-AVPNs.

(A3, A4 and B2, B3) Summarized

data for the frequency (A3, B2) and amplitude (A4, B3) of spontaneous EPSCs in IA-AVPN (A3, A4) and II-AVPNs (B2, B3). Note that SKF-86466 (10 mol·L-1) alone caused a significant frequency increase of spontaneous EPSCs, in both IA- and II-AVPNs. Mox, moxonidine. SKF, SKF-86466. *, P < 0.05; **, P < 0.01. Figure 3. The moxonidine-induced frequency decrease of spontaneous EPSCs

in AVPNs was attenuated by AGN-192403. (A1) Recoding of representative IA-AVPN (upper panel), along with integrated activity of hypoglossal activity (XII, lower panel). (A2) The recording segments in (A1) are shown in an enlarged scale, showing that after pretreatment with AGN-192403 (10 mol·L-1), moxonidine (10 mol·L-1) still induced a frequency decrease of spontaneous EPSCs in IA-AVPNs. (B1) Recording segments in a representative II-AVPN, showing that after pretreatment with AGN-192403 (10 mol·L-1), moxonidine (10 mol·L-1) induced a similar frequency decrease of spontaneous EPSCs as in IA-AVPNs. (A3, A4 and B2, B3) Summarized data for the frequency (A3, B2) and amplitude (A4, B3) of spontaneous EPSCs in IA-AVPN (A3, A4) and II-AVPNs (B2, B3). Note that AGN-192403 (10 mol·L-1) alone caused a significant frequency increase of spontaneous EPSCs, in both IA- and II-AVPNs. Mox, moxonidine. AGN, AGN-192403. *, P < 0.05; ***, P < 0.001. Figure 4. The moxonidine-induced frequency decrease of spontaneous EPSCs in AVPNs was abolished by efaroxan. (A1 and B1) Recoding of representative IA-AVPN (A1) and a representative II-AVPN (B1) (upper panels), along with integrated activity of hypoglossal activity (XII, lower panels). (A2 and B2) Recording segments in (A1) and (B1) are shown in an enlarged scale, showing that after pretreatment with efaroxan (10 mol·L-1), moxonidine (10 mol·L-1) did not cause any change of the spontaneous EPSCs in either IA-AVPNs (A2) or II-AVPNs (B2). (A3, A4 and B3, B4) Summarized data for the frequency (A3, B3) and amplitude (A4, B4) of spontaneous EPSCs in IA-AVPN (A3, A4) and II-AVPNs (B3, B4). Note that efaroxan (10 mol·L-1) alone did not cause any change of the spontaneous EPSCs in either IA-

and II-AVPNs. Mox, moxonidine. Efa, efaroxan

Highlights: 1. Moxonidine inhibits the excitatory inputs to airway vagal preganglionic neurons. 2. Selective 2-adrenoceptor antagonist partially blocked the effect of moxonidine. 3. Selective imidazoline I1 receptor antagonist partially blocked the effect of moxonidine. 4. Blocking 2-adrenoceptors and imidazoline I1 receptors abolished moxonidine effect.

5. Both 2-adrenoceptors and imidazoline I1 receptors regulate airway vagal activity.

Jijiang Wang designed the study and prepared the manuscript; Xujiao Zhou, Ding He, Xianxia Yan, Xingxin Chen, and Rui Li performed the experiments, analyzed the data and helped preparation of the manuscript; Guangming Zhang helped interpretation of the data and preparation of the manuscript.