Abstract
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.
Highlights
Bronchial asthma, a type of common chronic airway disease worldwide, has prominent features of airway hyper-responsiveness, inflammation and excessive activation of cholinergic fibers to the trachea and bronchioles
Inspiratory-activated airway vagal preganglionic neurons were first identified by the presence of fluorescence and by their characteristic distribution in the the external formation of the nucleus ambiguous (eNA), which is in the close ventral, ventrolateral and ventromedial vicinity of the the compact formation of the nucleus ambiguous (cNA) (Chen Y. et al, 2007; Chen et al, 2012a) (Figures 1A,B)
Inspiratory-activated airway vagal preganglionic neurons were defined as those that were activated during inspiratory phase and were manifested by the rhythmic inspiratory-related discharges under cell-attached configuration (Figure 1C), by the rhythmic bursts of excitatory postsynaptic currents (EPSCs) during inspiratory phase under voltage clamp recording (Figures 2A,D) or by the rhythmic inspiratory-related depolarizing excitatory postsynaptic potentials (EPSPs) superimposed by trains of action potentials under current clamp recording data not shown
Summary
A type of common chronic airway disease worldwide, has prominent features of airway hyper-responsiveness, inflammation and excessive activation of cholinergic fibers to the trachea and bronchioles. In reflex pathways the AVPNs located in medulla oblongata conveying signals from brain to the intrinsic tracheobronchial ganglia are considered to be very crucial for regulating airway function either in diseased conditions or normal states (Haxhiu et al, 2005; McGovern and Mazzone, 2010). AVPNs in the eNA function as the main neurons which modulate cholinergic tone of airway smooth muscles as well as control the intrapulmonary airway resistance and tracheobronchial caliber (Haselton et al, 1992; Canning and Fischer, 2001; Yan et al, 2017). AVPNs in the eNA exhibit different rhythmic changes of synaptic inputs in parallel with central inspiratory activities (Chen Y. et al, 2007; Qiu et al, 2011; Chen et al, 2012a,b; Abbreviations: 4V, 4th ventricle; ACSF, artificial cerebral spinal fluid; AMPA, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid; AP5, D-2-amino-5phosphonovalerate; CBX, carbenoxolone; AVPNs, airway vagal preganglionic neurons; cNA, the compact formation of the nucleus ambiguous; CNQX, 6-cyano7-nitroquinoxaline-2,3-dione; DHPG, (RS)-3,5-dihydroxyphenylglycine; DMSO, dimethyl sulfoxide; DMV, dorsal motor nucleus of vagus; eNA, the external formation of the nucleus ambiguous; EPSCs, excitatory postsynaptic currents; EPSPs, excitatory postsynaptic potentials; GABA, γ-aminobutyric acid; IA-AVPNs, inspiratory-activated airway vagal preganglionic neurons; ICSs, inward current spikelets; IO, inferior olive; INaP, persistent sodium currents; KATP, ATP-sensitive potassium channels; NA, nucleus ambiguous; NMDA, N-methyl-D-aspartate; PBC, pre-Bötzinger complex; PY, pyramidal tract; QX314, lidocaine N-ethyl bromide; ROb, raphe obscurus nucleus; NTS, nucleus tractus solitarius; sp, spinal trigeminal tract; Sp5I, spinal trigeminal nucleus, interpolar part; TBOA, DL-threoβ-benzyloxyaspartate; TRH, thyrotropin-releasing hormone; TTX, tetrodotoxin; XIIN, hypoglossal nucleus
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