Nicotinic Cholinergic Modulation Of The Airway Vagal Preganglionic Neurons And α₂-adrenergic Modulation Of Medullary Respiratory Rhythm Generator

Cholinergic and catecholaminergic mechanisms are involved in the physiological central control of cardiopulmonary functions, and play critical roles in the pathogenesis of some common respiratory disorders. This study investigates the cholinergic modulation of inspiratory-activated airway vagal preganglionic neurons (IA-AVPNs) and the a2-adrenergic modulation of medullary respiratory rhythm generator.The airway vagal preganglionic neurons (AVPNs) supply the essential excitatory drive to the postganglionic neurons and dominate the neural control of the airway physiologically and pathophysiologically. AVPNs express multiple subunits of nicotinic acetylcholine receptors (nAChRs), but the influences of exogenous nicotine and endogenous acetylcholine are unknown. In rhythmically firing medullary slices of newborn rats, this study examined the effects of nicotine and endogenous acetylcholine on retrogradely labeled, functionally identified IA-AVPNs using patch-clamp technique. The results are as follows:10μmol L-1nicotine significantly increased the frequency and amplitude of the spontaneous excitatory postsynaptic currents (EPSCs) of IA-AVPNs. These effects were insensitive to methyllycaconitine (MLA,100nmol L-1), an antagonist of α7type of nAChRs, but were prevented by dihydro-β-erythroidine (DHβE,3μmol L-1), an antagonist of α4β2type of nAChRs. Nicotine caused a tonic inward current in IA-AVPNs, which was reduced by MLA or DH(3E alone, but was not abolished by co-application of MLA and DHβE. Nicotine caused significant frequency increases in the spontaneous GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs), and significantly increased the amplitude of the spontaneous glycinergic IPSCs, all of which were prevented by DHβE. Nicotine had no effects on the miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mlPSCs) following pretreatment with TTX. Under current clamp, nicotine caused depolarization and increased the firing rate of IA-AVPNs during inspiratory intervals. Neostigmine (10μmol 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 the nicotinic effects are mediated presynaptically by activation of α4β2type of nAChRs and postsynaptically by activation of multiple nAChRs including α7and α4β2types. The innovation points of this part of the research:this is the first study that examines the effects of nicotine and neostigmine on the synaptic inputs of retrogradely labeled, functionally identified tracheobronchial-projecting IA-AVPNs, which demonstrates that the nAChRs located on both the glutamatergic and the GABAergic/glycinergic neurons preceding the IA-AVPNs are of a4β2type.From the moment of birth, the respiratory rhythm generator (RRG) of mammals should be able to elaborate its rhythmic command and adjust it to behavioral and environmental changes. It has been well established that RRG is located in the rostral ventrolateral medulla (RVLM) and the pre-Botzinger complex (PBC). RRG activity is regulated by the central endogenous adrenergic-noradrenergic systems. Previous studies indicate that the central endogenous adrenergic-noradrenergic systems facilitate RRG activity via activation of α1-adrenoceptors but inhibit it via a2-adrenoceptors. It is unclear how α1-ARs and α2-ARS regulate RRG activity at the synaptic level. Agonists of α2-ARs are commonly used clinically in the induction and maintenance of anesthesia and analgesia, and in the treatment of hypertensive diseases. This study is mainly concerns the α2-adrenergic modulation of medullary RRG. In addition, since most agonists of α2-ARS have binding affinity toⅢ-Rs, we also investigated the modulation of medullary RRG by Ⅲ-Rs.In medullary slices of newborn rats with respiratory-like hypoglossal rhythm, this study investigated the effects of agonists of α2-ARS on RRG activity using recording of hypoglossal rootlets; and the effects of moxonidine, an agonist of α2-ARs with potent binding affinity to Ⅲ-Rs, were studied at synaptic levels using patch-clamp recording of RRG respiratory neurons. In anesthetized adult rats, the respiratory effects of intracisternal moxonidine were studied using plethysmography. The results show that all of the agonists of α2-ARs tested, with or without binding affinity to Ⅲ-Rs, consistently caused a significant frequency decrease of the hypoglossal bursts; and moxonidine (10μmol L-1) significantly inhibited the tonic glutamatergic inputs of respiratory neurons in the pre-Botzinger complex. The moxonidine-induced changes in vitro were only partially blocked by selective antagonist of α2-ARs SKF-86466(10μmol L-1) or by adenylyl cyclase activator forskolin (10μmol L-1), but enhanced by cAMP-dependent protein kinase inhibitor H-89(10μmol L-1). However, these changes were completely prevented by efaroxan (10μmol L-1), an antagonist of α2-ARS and Ⅲ-1Rs. In vivo, intracisternal moxonidine (100nmol) induced typical tidal breathing, along with a progressive decrease in respiratory frequency and an increase in tidal volume, all of which were insensitive to SKF-86466(200nmol) but completely prevented by efaroxan (200nmol). These results demonstrate that Ⅲ-1Rs are involved in physiological RRG control, and suggest that over activation of Ⅲ-1Rs and/or α2-ARs inhibits the RRG, which might involes inhibition of the glutamatergic neurotransmission through which the RRG generate and maintain respiratory rhythm.The innovation points of this part:the results of this study demonstratethat Ⅲ-Rs are involved in RRG regulation, and suggest that activation of Ⅲ-Rs, alone or together with a2-ARs, causes respiratory inhibition, and inhibits the excitatory synaptic inputs of respiratory neurons of RRG in vitro; and induces Cheyne-Stokes respiration in vivo.