Regulation Of Breathing By Phox2b-expressing Neurons In The Locus Coeruleus

Sleep-related disordered breathing(SRDB)is a group of diseases characterized by repeated apnea or hypoventilation during sleep.SRDB consists of obstructive sleep apnea,sleep-related alveolar hypoventilation and central sleep apnea.Congenital central hypoventilation syndrome(CCHS)is a rare genetic disease characterized by dysfuncational autonomic breathing.The paired-like homeobox 2b(Phox2b)gene mutation is considered the key cause of CCHS,according to the guidelines issued by the American Thoracic Society.Phox2b is a paired homeobox gene located on the 4^th chromosome.It codes for a homeodomain transcription factor involved in the development of the autonomic nervous system during embryonic development.This transcription factor is expressed primarily in neurons that control respiratory and cardiovascular reflexes,including the solitary tract nucleus(NTS),retrotrapezoid nucleus(RTN)and locus coeruleus(LC),where these neurons serve as central respiratory chemoreceptors.Accumulated evidence has shown that selective activation of Phox2b neurons in RTN or NTS potentiates central respiratory drive;Selective ablation of these neurons attenuates a hypercapnic ventilatory response.However,it remains to explore whether Phox2b^LCneurons contribute to regulation of breathing.LC,located in the posterior area of the rostral pons in the lateral floor of the fourth ventricle,participates in regulation of many physiological functions.The majority of LC neurons are noradrenergic and LC is the main source of noradrenergic fiber projection in the central nervous system.Although prior studies have shown that the destruction of catecholaminergic LC neurons could inhibit hypercapnia ventilatory responses.But,the effect of Phox2b^LCneuron activation on resting breathing remains to be further studied.It also has been reported that the number of LC chemosensitive neurons decrease with age.The chemosensitivity of LC neurons is the basis of contribution to central respiratory reflex.Therefore,whether LC neurons still retain pH sensitivity in adult animals is of importance.LC neuronal axons project extensively to the brain regions that regulate the autonomic nervous system and also receive innervations from NTS,RTN,parabranchial nucleus(PBN)and pre B(?)tzinger Complex(preB(?)tC).However,the circuit mechanism responsible for regulation of breathing by LC neurons is little understood.Phox2b regulates the differentiation of embryonic noradrenergic neurons and affects the phenotype of noradrenergic neurons.LC neuron are absent in both CCHS patients and animal models with Phox2b mutation.However,whether the Phox2b^LC neurons play a role in the pathogenisis of CCHS is not clear.Therefore,it is of great clinical significance to investigate the effects of Phox2b^LC neurons on resting breathing and central respiratory chemoreceptor reflex.Taken together,the present study aimed to investigate the role of Phox2b^LC neurons in controlling breathing and possible circuit mechanisms.The project will help better understand the pathogenesis of CCHS and other sleep-disordered breathing disorders,and provides new clues for precise intervention of these diseases.Objective:To study the role of Phox2b^LC neurons in controlling breathing and possible circuit mechanisms.To that end,we will study:(1)the effect of selective activation of Phox2b^LC neurons on resting breathing;(2)the effect of selective ablation of Phox2b^LC neurons on hypercapnia ventilatory response;(3)CO₂ sensitivity of Phox2b^LC neurons in adult mice;(4)Analysis of anatomical and functional relstionship between Phox2b^LC neurons and respiratory central pattern generators.Methods:Adult male Phox2b-Cre and Phox2b-EGFP transgene mice were used in the experiment.(1)The lung function was measured using a Whole Body Plethysmography system(WBP).Breathing parameters consist of respiratory frequency(RF),tidal volume(TV)and minute ventilation(MV).(2)The chemogenetic technique,achieved by microinjections of Cre-inducible vectors encoding a Gq-coupled human M3 muscarinic receptor(h M3Dq)into LC,was employed to selectively activate Phox2b^LC neurons in conscious mice to assess changes in breathing parameters.(3)The optogenetic technique,achieved by microinjections of Cre-inducible vectors encoding light-sensitive channelrodopsin type 2(Ch R2)into LC,was used to activate Phox2b^LCneurons in anesthetized mice and phrenic nerve discharge was used as an indicator of central drives to breathing.(4)Genetical apoptosis of Phox2b^LCneurons was used to evaluate changes in the hypercapnia ventilatory response.(5)The CO₂ sensitivity of Phox2b^LC neurons was assessed using immunohistochemistry(c Fos).(6)Anatomical connections and functional links between Phox2b^LC neurons and respiratory central pattern generators were assessed using genetic techniques and neuronal tracing techniques.Result:1.Immunofluorescence results showed that the majority of EGFP⁺neurons from LC in Phox2b-EGFP adult mice were TH⁺and Phox2b⁺;Phox2b⁺TH⁺neurons accounted for>50%of the total number of TH⁺neurons,and accounted for>52%of the total number of Phox2b⁺neurons.Moreover,the distribution of Phox2b⁺neurons and TH⁺neurons was different.Phox2b⁺TH⁺neurons were distributed mainly at the caudal LC and sparcely at the rostral part.The results of PCR also confirmed the presence of Phox2b in LC in adult mice.2.Immunofluorescence results indicated that Phox2b⁺mCherry⁺neurons accounted for 92%of the total number of transduced mCherry⁺neurons,suggesting that majority of the neurons activated by chemogenetics or optogenetics were Phox2b^LCneurons.3.Chemogenetic stimulation of Phox2b^LC neurons produced a robust long-lasting increase in RF and MV,with a relatively slow onset,long-lasting effect(>120 minutes).At the same time,inspiratory time was reduced and TV were unchanged significantly.4.In anesthetized mice with mechanical ventilation and vagal nerves-cutoff,under the condition of end-tidal CO₂=4%,optogenetic stimulation of Phox2b^LC neurons increased moderately the frequency of phrenic nerve discharge(inspiratory frequency),without significant change in amplitude,in favor of enhanced central drive to breathing.5.Selective ablation of Phox2b^LC neurons(mRNA level of Phox2b decreased by 40%)reduced considerably TV and MV during exposure to 5%CO₂ when compared to controls.During exposre to 8%CO₂,RF,TV and MV in mice with loss of Phox2b^LC neurons were less than those in control mice,indicating that loss of a certain proportion of Phox2b^LCneurons attenuated the hypercapnic ventilatory response.6.In response to inhaling 8%CO₂,17%of Phox2b^LC neurons were activated,as well as 18%of cFos⁺neurons were Phox2b⁺.Therefore,a subset of Phox2b^LC neurons were CO₂-sensitive.This type of neuron may be the key componet for the hypercapnia ventilatory response.7.Selective stimulation of Phox2b^LC neurons significantly activated preB(?)tC neurons,suggesting that activation of Phox2b^LC neurons affected breathing probably via activation of preB(?)tC neurons.8.Neuronal tracing studies have found that the axons of Phox2b^LCneurons directly projected to preB(?)tC and PBN,suggesting that Phox2b^LCneurons may controlling breathing by direct effect on the activity of the respiratory central pattern generator through monosynaptic connections.Conclusion:Selective activation of Phox2b^LC neurons potentiates central respiratory drive and therefore increase resting pulmonary ventilation;Selective ablation of these neurons attenuates the hypercapnic ventilatory response.The aformentioned effects are most likely achieved by direct regulation of respiratory central pattern generator via monosynaptic connections.