Circuit Mechanism Underlying Contribution Of Central Respiratory Chemoreceptors To Synergistical Control Of Breathing

Congenital central hypoventilation syndrome（CCHS）is a rare genetic respiratory disorder characterized by the reduced or absent ventilatory responses to hypercapnia and hypoxemia during sleep.CCHS is an autosomal dominant disorder,and mutations in the paired-homobox 2b gene（paired-like homeobox 2b,Phox2b）leading to polyalanine repeat amplification are a key causative factor in CCHS.The transcript factor Phox2b is restrictively expressed in central respiratory chemoreceptors in brainstem,including nucleus tractus Solitarii（NTS）,retrotrapezoid nucleus（RTN）,and locus coeruleus（LC）.Central respiratory drive force originates from the pre B（?）tzinger complex（pre B（?）t C）in medullary ventral respiratory group.Central respiratory chemoreceptors provide excitatory drive to the pre B（?）t C to subsequently induce adaptive ventilatory response.However,the synergistic regulation of respiration between the central respiratory chemoreceptor neurons of NTS,RTN and LC expressing Phox2b and the functional loop mechanism with pre B（?）t C remain to be further revealed.Objective:This study aims to reveal the circuit mechanism of central respiratory chemoreceptors regulating respiration.The specific contents included:（1）to observe the effects of selective activation or inhibition of pre B（?）t C-projecting Phox2b neurons on respiratory function in mice,（2）to clarify the effects of selective activation or inhibition of pre B（?）t C-projecting Phox2b neurons on apnea and sigh in mice,（3）to reveal the anatomical projections of between Phox2b-expressing NTS,RTN and LC neurons or with pre B（?）t C.Methods:1.Adult male Phox2b-Cre mice（25～30 g）were used in the experiments.2.Retrograde tracer techniques were used to reveal the pre B（?）t C-projecting Phox2b neurons in the NTS,RTN and LC.3.Lung function test was performed in conscious mice using a whole-body plethysmography system.Breathing parameters consist of breathing frequency（BF）,total volume（TV）,minute ventilation（MV）,apnea and sigh.4.Chemogenetics was performed to activate or inhibit pre B（?）t C-projecting Phox2b neurons in Phox2b-Cre mice to evaluate the changes of-breathing parameters.5.Using anterograde or retrograde neural tracer technique to reveal the mutual projections of Phox2b-expressing neurons among NTS,RTN and LC.Results:1.Immunofluorescence results revealed that Phox2b-expressing NTS(NTS^Phox2b),RTN(RTN^Phox2b)and LC(LC^Phox2b)neurons innervated pre B（?）t C.2.Chemogenetic activation of pre B（?）t C-projecting Phox2b neurons increases BF and MV in the long-term,decreases the number of apnea and the cumulative duration of apnea,and increases the number of sighing.In addition,activation of pre B（?）t C-projecting Phox2b neurons enhance central respiratory chemoreceptors reflex（hypercapnic ventilatory response）.3.Chemogenetic inhibition of pre B（?）t C-projecting Phox2b neurons shows no significant changes in BF,TV and MV,and no significant changes in the number of apnea,the cumulative duration of apnea and the number of sighing.In addition,inhibition of pre B（?）t C-projecting Phox2b neurons enhance central has no effect on respiratory chemoreceptors reflex.4.The neural tracer results demonstrate that the axons of NTS^Phox2bneurons project to RTN and LC,and the axons of RTN^Phox2b neurons project to NTS and LC,while the axons of LC^Phox2b neurons did not innervate NTS and RTN,revealing the existence of neural circuits between central respiratory chemoreceptors.Conclusions:This study revealed at the neural circuit level that simultaneous activation of pre B（?）t C-projecting Phox2b neurons in the NTS,RTN,and LC increased basal lung ventilation and sigh,reduced the occurrence of apnea,and increased the sensitivity of responded to hypercapnic ventilatory response.These effects are likely to be synergistic regulated by central respiratory chemoreceptors and/or between central respiratory chemoreceptors and pre B（?）t C.