Effects And Mechansim Of Thyrotropin-releasing Hormone On Airway-related Vagal Preganglionic Neurons

Airway-related vagal preganglionic neurons (AVPNs) are primarily located in three sites in the medulla:the compact portion of the nucleus ambiguus (cNA), the external formation of the nucleus ambiguus (eNA) and the dorsal motor nucleus of the vagus (DMV). Laryngeal nerves, which arise from neurons in cNA, play an important role in controlling breathing and vocalizing. AVPNs in DMV predominantly project to tracheobronchial secretory glands. However, AVPNs in eNA have an important role in controlling airway resistance. AVPNs in eNA include inspiratory-activated airway-related vagal preganglionic neurons (IA-AVPNs) and inspiratory-inhibited airway-related vagal preganglionic neurons (Ⅱ-AVPNs). AVPNs receive excitatory inputs and inhibitory inputs, such as glutamatergic inputs, GABAergic inputs, glycinergic inputs and so on. In addition, some neurotransmitters or neuromodulators such as norepinephrine, acetylcholine and serotonin modulate the activity of AVPNs. The AVPNs receive dense inputs from terminals containing thyrotropin-releasing hormone (TRH). And TRH microinjection into the nucleus ambiguus (NA) caused slight dilation followed by constriction of the tracheal smooth muscles. However, central distributions and the electrophysiological properties of AVPNs are unclear at present. In addition, how TRH regulates AVPNs at synaptic level remains unknown. In the present study, using retrogradely fluorescent labeling and patch clamp techniques, central distributions and the electrophysiological properties of the AVPNs were observed in medullary slices from2-6-day-old Sprague-Dawley rats. In addition, the effects and mechanism of TRH on AVPNs were further studied. The study will give some clues for central mechanisms of clinical diseases such as asthma, chronic obstructive pulmonary diseases, and obstructive sleep apnea syndrome. Results and conclusions are as follows:1. The electrophysiological properties and modulation andof AVPNs(1) The electrophysiological properties of AVPNsThe AVPNs in the eNA were identified in rhythmically firing brainstem slices using retrogradely fluorescent labeling techiniques. It is demonstrated that IA-AVPNs and II-AVPNs have different distributions in the medulla. In a collection of129fluorescence-labeled AVPNs,64.3%are inspiratory-activated and85.6%of the IA-AVPNs are located in the close ventrolateral vicinity,14.4%in the close ventral or ventromedial vicinity, of the cNA.35.7%of the labeled AVPNs are inspiratory-inhibited.89.1%of the Ⅱ-AVPNs are located in the close ventral or ventromedial vicinity, and10.9%are in the ventrolateral vicinity of the cNA.IA-AVPNs showed inspiratory-related augmentation of the EPSCs, but II-AVPNs showed inspiratory-related augmentation of the IPSCs. Under current clamp, Ⅱ-AVPNs exhibit significantly more positive resting membrane potential, more negative voltage threshold and lower minimal current required to evoke an action potential. The afterhyperpolarization in IA-AVPNs is mediated by small-conductance Ca2+-activated channels; and that the afterhyperpolarization in II-AVPNs is mediated by large-conductance Ca2+-activated 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. Within the voltage range of-20to+30mV, the evoked long lasting outward current was significantly inhibited by4-AP in inspiratory-activated AVPNs, but was unaltered in inspiratory-inhibited AVPNs. These results suggest that IA-AVPNs and Ⅱ-AVPNs have different central distribution and express different types of voltage-gated ion channels.(2) The modulation of Ⅱ-AVPNs by TRH and the mechanisms100nM TRH significantly enhanced both the frequency and amplitude of hypoglossal bursts, and caused a tonic excitatory inward current of50.1±7.4pA in II-AVPNs at a holding voltage of-80mV. In the presence of TTX, TRH caused a tonic inward current of32.1±4.7pA, suggesting that voltage-gated sodium channels contributed partly to the genesis of the inward current. The frequency of the spontaneous excitatory postsynaptic currents (EPSCs) and miniature excitatory postsynaptic currents (mEPSCs) in the Ⅱ-AVPNs, was not significantly changed by TRH. At a holding voltage of-50mV, the Ⅱ-AVPNs showed both spontaneous and phasic inspiratory inhibitory postsynaptic currents (IPSCs). In most Ⅱ-AVPNs, the IPSCs during inspiratory intervals were scarce and the phasic inspiratory inhibitory currents were blocked by focal application of strychnine. TRH had no effect on the spontaneous IPSCs but significantly attenuated the phasic inspiratory outward currents, in both amplitude and aera. 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 excites the Ⅱ-AVPNs both postsynaptically via a direct excitatory current and presynaptically via attenuation of the phasic glycinergic synaptic inputs.2. The modulation of IA-AVPNs by TRH and the mechanismsUnder voltage clamp,100nM TRH caused a tonic excitatory inward current of IA-AVPNs, enhanced the excitatory inputs and evoked a distinct oscillatory pattern of the baseline current. The slow excitatory inward current and the oscillatory pattern were unaffected by blockade of chemical synapses. Gap junction blocker carbenoxolone (100μM) prevented the oscillatory pattern without affecting the slow excitatory inward current and the enhancement of the excitatory inputs. Tetrodotoxin, a blocker of both the voltage-dependent sodium transient and persistent sodium current, riluzole, a blocker of the persistent the sodium current, and cadmium chloride, a non-selective blocker of the voltage-dependent calcium channels, each blocked a major proportion of the slow excitatory inward current and prevented the oscillatory pattern. Under current clamp TRH caused a slow depolarization and continuous or oscillatory firing. These results demonstrate that TRH excited the IA-AVPNs presynaptically via enhancement of the excitatory inputs, and postsynaptically via a slow excitatory inward current and a gap junction-mediated oscillatory pattern.