Regulation Of Breathing By Orexin Signaling Pathway In Nucleus Tractus Solitarii

Respiratory chemoreflexes is a pivotal homeostatic mechanism to maintain acid-base balance by excretion of excessive CO₂.Regulaiton of breathing（e.g.respiratory rate and amplitude）is mainly achieved by neural network organized within serially arrayed and functionally interacting brainstem compartments extending from the pons to the lower medulla.The Botzinger complex and the pre-Botzinger complex are the core circuit components that produce respiratory rhythm.Central chemoreceptors and pons provide excitatory drives for rhythm center and central pattern generators.The nucleus tractus solitarii（NTS）is the primary integration center of cardiovascular and respiratory afferent information and is also an important central respiratory chemoreceptor candidate.Although the NTS neurons project their axons to the ventrolateral respiratory column and affect rhythmic neurons through synaptic connections,the architecture of neural circuits involved in the projections remain poor understood and the neurochemical phenotype of projection neurons remains to be examined.Orexin is synthesized and secreted by hypothalamic orexin neurons and contributes to regulation of multiple physiological functions,including energy metabolism,feeding behavior,the alternating of sleep-wake,rewarding and so on.Orexin plays an important part in central control of breathing.It has been resported that the orexin signaling pathway,located in pre-Botzinger complex,rostral ventrolateral medulla,Kolliker-Fuse nucleus,hypoglossal nucleus,medullary raphe and retrotrapezoid nucleus,all contributes to regulation of respiratory frequency and amplitude.The change in respiratory pattern is depedent on neuronal projection pathway,animal state（wakefulness or sleep）and circardian and so on.Orexin receptors have been found to be present in the NTS.However,it remains unknown whether the orexin signaling pathway contributes to controlling breathing in the NTS.Objective: To examine the role of orexin in controlling breathing in the NTS.Methods: The viral vector AAV-U6-OX1R-shRNA-EGFP was injected into the rat NTS to knock down expression of OX1Rs（called “OX1R-shRNA group”）,as well as AAV-U6-scrambled-shRNA-EGFP for control.Immunofluorescence technique was used to reveal expression of EGFP.The inhibition efficiency of OX1 R was determined by qRT-PCR.Plethysmography was applied to examine effect of inhibiting OX1 R on hypercapnic ventilatory response in conscious rats,as reflexed by tidal volumn（TV）,breathing frequency（BF）and minite ventilation（MV）.The phrenic nerve discharge,blood pressure and heart rate were recorded in anesthetized rats in order to test effects of orexin on cardiopulmonary activity.Results:1 The immunofluorescence results reveal the presence of OX1 R and expression of EGFP in the NTS.2 OX1 R mRNA is reduced by ¹5% in the OX1R-shRNA group compared with control.3 During exposure to 0% CO₂（balance by 100%O₂）,no significant difference is found in breathing parameters between two group,with the exception that BF is far lower in OX1R-shRNA group compared with counterparts（P<0.0001）.4 During stepwise increases in concentration of CO₂（2-8%）in inspired air,TV,BF and MV were all raised in two groups of rats,with a higher increase in TV（P<0.05⁰.0001）,and a much lower increase in BF and MV in the OX1R-shRNA group in relative to control group（P<0.05⁰.0001）.5 Microinjection of OrexinA into the NTS reduced the frequency（P<0.0001）but not amplitude of phrenic nerve discharge,as well as blood pressure（P<0.001）and heart rate（P<0.01）in anesthetized rats.Conclusions: The reduction of NTS OX1 Rs attenuates hypercapnic ventilatory response in conscious rats,whereas activation of OX1 Rs reduces prenic nerve activity in anesthetized rats.Regulation of breathing by orexin is most likely state-dependent in the NTS.