5-HT→5-HT_2AR→PKC Pathway In Mediating Phrenic Longterm Facilication In Rats

【Background】Respiration, a spontaneous rhythmic motor and one of the basiclife behaviors in maintaining body metabolism and function, varys with internaland external circumstances of each individual. The ability that alters respiratorymotor output to accommodate the ever-changing needs of the individual isregarded as respiratory plasticity. Failure of respiratory plasticity often leads tomalfunctions, such as sudden infant death syndrome and obstructive sleepapnea syndrome . Considering the tight relationship of respiratory plasticity withdiseases, people have paid much attention to the mechanism underlyingrespiratory plasticity, especially under pathological state. To date, the mostfrequently studied model of respiratory plasticity is long-term facilitation (LTF)model. LTF is a type of neuronal plasticity characterized by a progressiveincrease in phrenic nerve activity that lasts for at least one hour after stimuli(repetitive hypoxia, drug exposure, etc.), resulting in the increased pulmonaryventilation. LTF expression will help stabilize upper airways and maintain airwaypatency, implicating a potential clinical promise in treatment of sleep disorders. Phrenic LTF (pLTF) is usually induced by repetitive hypoxia in vivo, orrepetitive 5-HT application in vitro slice preparation. 5-HT/5-HT2A receptor (5-HT2AR) system is necessary and sufficient for pLTF expression, andabnormalities of this system often lead to respiratory disorders. However, thedownstream intracellular signalings activated by 5-HT system are little known sofar. In the present study, we established a novel pLTF model through eposodichypobaric hypoxia to mimik a high-altitude hypoxic situation. Additionally, wefound that a bolus of systemic 5-HT admistration could exert the enhancedphrenic nerve activity and pLTF, which was proviously uncharacterized. Based onour established two pLTF models in combination with drugs'intervention, theunderlying intracellular signalings were then investigated in the present study.【PurposPurpose】Using eposodic hypobaric- and 5-HT-induced pLTF models, we aimedto explore the roles of 5-HT→5-HT2AR→PKC pathway in respiratoryneuroplasticity in rats.【Methods】(1) Rats were housed in a chamber and maintained alternately under 5min of hypobaric hypoxia and 5 min of normoxia between for 7 consecutive days.Hypobaric hypoxia was achieved by continuous air evacuation to gradually reacha high altitude of about 9000～10000 m. On the morning of the eighth day,phrenic nerve activity was recorded after three episodic hypoxia. (2) Animalswere anaesthetized intraperitoneally and subjected to bilateral midcervicalvagotomy and paralyzed with intraperitoneal injection of curarine to preventspontaneous breathing effort. A femoral arterial catheter was placed to allowblood sample withdrawal for blood gases and blood pressure measurement. Afemoral venous catheter was implanted for drugs and fluid administration. Theright phrenic nerve was isolated unilaterally and recorded after drugsadministration or episodic hypoxia. 【ResultResults】An augmented pLTF lasting for at least 60 min after AIH stimulus wasidentified in CIH-pretreated rats, which was almost doubled in comparison withcontrol animals (receiving AIH alone). Ketanserin, a selective 5-HT2ARantagonist, partly blocked the augmented pLTF induced by CIH. PKCθinhibitorcompletely blocked pLTF, and led to irregular patterns of phrenic nerve activity inCIH rats.An immediate inhibition of phrenic nerve activity was consistentlydetectable in all animals with systemic 5-HT exposure, though the extent to whichdifferent doses of a bolus of 5-HT exerted was different. A bolus of 5-HTapplication at 20μg/kg dose elicited a slight inhibition of phrenic nerve activity,which returned to baseline immediately. 5-HT at 40μg/kg caused a transientinhibition in both the amplitude and the frequency. 5-HT at 100μg/kg dose gaverise to an immediate, marked inhibition and a subsequent striking facilitation ofphrenic nerve activity. The enhanced phrenic nerve activity could last for at least60 min after 5-HT pretreatment, representing the characteristic of pLTF.Ketanserin completely blocked 5-HT-induced pLTF, whereas WAY100635, aselective 5-HT1AR antagonist, didn't work. PKC inhibitor, staurosporine, didn'taffect the initial inhibition induced by 5-HT, but substantially inhibited thesubsequent facilitation, leading to a complete blockage of pLTF. We then foundthat 5-HT-induced biphasic pattern formation and pLTF expression werecontributed by two separate mechanisms, the initial inhibition in association withthe nodose ganglion, and the subsequent brisk facilitation with the carotid body.Hence, 5-HT led to an enhanced pLTF when the initial inhibition was eliminatedwith bilateral nodose ganglionectomy. On the other hand, with bilateral carotidbody excision, the subsequent robust facilitation was abolished, and also the pLTF.【Conclusions】Continuous sensory afferent inputs, central signal integration, and phrenic motor output constitute the complex respiratory network. pLTFexpression no doubt needs entire respiratory network activity. Indeed, the presentstudy unraveled a peripheral dynamic balance between carotid body excitationand nodose ganglion inhibition that contributed critically to 5-HT-induced pLTFexpression. 5-HT→5-HT2AR→PKC pathway plays an important role in pLTFformation, which is evident in both our established hypobaric- and 5-HT-inducedpLTF rat models. Our findings will extend our understanding of the mechanismundertaking LTF expression, and provide the potential likelihood for clinicaltreatment of sleep disorders.