Regulation Of Breathing By Phox2b-expressing Neurons In The Nucleus Tractus Solitarii

Sleep-related breathing disorders are characterized by abnormal breathing during sleep,including obstructive sleep apnea,central sleep apnea,and sleep-related alveolar hypoventilation.Among them,congenital central hypoventilation syndrome（CCHS）is a rare hereditary sleep-disordered breathing manifesting severe alveolar hypoventilation frequently during sleep and requiring mechanical ventilation.When alveolar hypoventilation occurs,the sensitivity to hypercapnic and hypoxic responses is considerably reduced or even lost,eventually causing respiratory failure.Gene sequencing evidence shows that more than 90%of CCHS patients exhibit paired-like homeobox 2b（Phox2b）mutations.The Phox2b is located on the fourth pair of autosomes and contributes to the differentiation and development of the embryonic autonomic nervous system.The Phox2b is expressed on respiratory afferent pathways,including the carotid body（CB）,nucleus tractus solitarii（NTS）,retrotrapezoid nucleus（RTN）,and locus coeruleus（LC）.The central respiratory chemoreflex is an important homeostatic mechanism by sensing changes of CO₂/H⁺in the extracellular fluid,excreting excessive CO₂ and regulating lung ventilatory responses,ultimately maintaining the body’s acid-base balance.As the central respiratory chemoreceptor,at least the following criteria need to be met:（1）Intrinsic sensitivity to changes of CO₂/H⁺within physiological range;（2）In vivo CO₂stimulation induces adaptive ventilation;（3）The synaptic connectivity with the respiratory rhythm center so that selective activation,inhibition or destruction of this chemoreceptor population can facilitate or inhibit respiratory chemoreflex.In conclusion,the central respiratory chemoreceptors provide an excitatory drive for the respiratory central pattern generators to regulate ventilation responses.In case of dysfunction of these cells,it attenuates the central respiratory drive,ultimately resulting in hypoventilation or apnea.Prior studies demonstrated that selective activation of Phox2b-expressing RTN neurons enhanced central respiratory drive and destruction of Phox2b neurons attenuated respiratory chemoreflex.Furthermore,TASK-2 channels and G protein coupled receptor 4（GPR4）mediate the pH sensitivity of RTN neurons.Therefore,Phox2b-expressing RTN neurons serve as central respiratory chemoreceptors.Early studies found that NTS neurons are involved in respiratory regulation and some neurons display robust pH sensitivity.However,the neurochemical phenotype of those NTS neurons as central respiratory chemoreceptors remained to be addressed,and the pH-sensitive molecular substrates of these NTS neurons were yet to be further revealed.In summary,the present study attempts to investigate whether Phox2b-expressing NTS neurons are involved in respiratory control and the possible mechanism involved.To that end,experiments would be carried out to address:（1）effects of activation of Phox2b neurons on breathing;（2）impact of Phox2b neuron loss on central respiratory chemoreflex（hypercapnic ventilatory response）;（3）Whether GPR4 signaling mediates central respiratory chemoreflex in the NTS.PARTⅠ:Selective stimulation of Phox2b-expressing NTS neurons potentiates breathingObjective:To examine the effect of stimulation of Phox2b neurons on breathing.Methods:Experiments were performed in the Phox2b-Cre transgenic mouse line.The NTS was microinjected by saline（control）and cre recombinase-depedent vectors,including AAV-EF1α-DIO-mCherry（control）,AAV-EF1α-DIO-hM3Dq-mCherry（chemogenetics）and AAV-EF1α-DIO-Ch R2-mCherry（optogenetics）,respectively.After 4 weeks,hM3Dq-mCherry or ChR2-mCherry was successfully expressed in Phox2b neurons.The effect of Phox2b neuron activation on pulmonary ventilation in awake mice was examined by the chemogenetic approach,so was the action of photostimulation of Phox2b neurons on the phrenic nerve discharge（PND）in anesthetized mice using optogenetics.Whole body plethysmography（WBP）was used to determine tidal volume（TV）,respiratory frequency and minute ventilation（MV）.The radio telemetry system was applied to monitor systolic blood pressure（SBP）,diastolic blood pressure（DBP）,mean arterial pressure（MAP）and heart rate（HR）.Results:1.Immunofluorescence results showed that Phox2b⁺mCherry⁺neurons accounted for⁸3%of the total number of mCherry⁺neurons and for 13%of Phox2b⁺neurons.Cell-attached recordings in brainstem slices indicated that bath application of Clozapine N-oxide（CNO）depolarized hM3Dq-expressing Phox2b neurons.The results suggested that CNO activated the majority of hM3Dq-transduced Phox2b neurons.2.Compared with the control,chemogenetic activation of Phox2b neurons increased respiratory frequency and MV for a long period of time（>90 minutes）,with insignificant change in TV.3.Activation of Phox2b neurons reduced SBP,DBP and MAP 30 minutes after CNO injections.HR significantly increased 30 minutes after CNO injections and then gradually restored to the control level.Therefore,chemogenetic stimulation of Phox2b neurons reduced moderately blood pressure.4.The combined application of CNO and 5%CO₂ significantly increased respiratory frequency and MV except for TV than that treated with 5%CO₂ or CNO alone.Overall,stimulation of Phox2b-containing neurons reforced the central respiratory chemoreflex.5.In anesthetized,vagotomized and mechanically ventilated mice,photostimulation of Phox2b neurons quickly elicited recurrent PND during apnea（end-tidal CO₂ concentration（ETCO₂）=1%）.Moreover,the frequency but not amplitude of PND significantly increased when ETCO₂ was kept at3%.Therefore photostimulation of Phox2b-expressing NTS neurons enhanced a respiratory drive in anesthetized mice.6.Immunofluorescence results indicated that cFos⁺mCherry⁺neurons produced by 8%CO₂ stimulation accounted for 18%of the total number of mCherry⁺neurons and for 12%of the total number of cFos⁺neurons.Therefore,a subset of Phox2b neurons are CO₂-activated.7.Neuronal tracing experiments demonstrated that the axons from Phox2b neurons projected directly to the ventrolateral medulla,probably including the rostral ventral respiratory group（rVRG）,pre-B?tzinger complex and B?tzinger complex.Summary:Chemogenetic or optogenetic activation of Phox2b neurons strengthens pulmonary ventilation or central respiratory drive;the anatomical basis is monosynaptic connectivity between Phox2b-containg NTS neurons and ventrolateral medulla.The regulatory mechanisms involve the activation of both CO₂-sensitive and CO₂-insensitive neurons.PARTⅡ:Lesion of Phox2b-expressing NTS neurons attenuated a hypercapnic ventilatory responseObjective:To determine the impact of lesions of Phox2b-expressing NTS neurons on a hypercapnic ventilatory response in conscious miceMethods:Experiments were conducted in adult male ICR mice.The animals were divided into three groups by injections of saline,Blank-saporin（SAP）and substance P-saporin（SSP-SAP）.The neurotoxin SSP-SAP was used for lesions of Phox2b neurons.WBP was adopted to record respiratory parameters in awake mice.Results:1.When inhaled 0-8%CO₂,there were no noticeable difference in TV,respiratory frequency and MV between carotid body innervated and denervated mice,suggesting that the hypercapnic ventilatory response was mainly mediated by central but not peripheral respiratory chemoreceptors.2.MV and TV were both reduced by 13%during exposure to 8%CO₂ in inspired air when¹3%of the Phox2b-expressing neurons were eliminated.However,a loss of¹8%of these neurons was associated with considerable decreases in MV by≥18%and in TV by≥22%when challenged by≥4%CO₂.In both cases,breathing frequency was insignificantly different between groups.Moreover,destruction of Phox2b neurons does not affect basal respiratory parameters.3.Immunofluorescence results indicated that Phox2b is expressed in⁷4%of CO₂-sensitive neurons,whereas 10%of Phox2b neurons were CO₂-sensitive neurons.Summary:Destruction of Phox2b-expressing NTS neurons attenuated central respiratory chemoreflex,which was ascribed to a significant reduction in the number of CO₂-sensitive Phox2b neurons.PARTⅢInvolvement of NTS neuronal GPR4 in the hypercapnic ventilatory responsesObjective:To determine effect of GPR4 knockdown on hypercapnic ventilatory responses in the NTSMethods:Experiments were carried out in ICR adult male mice and they were divided into two groups:Scrambled-shRNA and GPR4-shRNA.The mice were injected with Ad-GPR4-shRNA-EGFP into the NTS for GPR4knockdown to assess whether the hypercapnic ventilatory response was attenuated.Western Blot and q PCR analysis were used to determine the knockdown efficiency of GPR4.The number of CO₂-sensitive neurons was calculated by immunofluorescence staining.Results:1.In the GPR4-shRNA group,the GPR4 mRNA level was reduced approximately by 30%and the protein content was reduced approximately by22%.2.Inhalation of 4-8%CO₂ resulted in a significant decrease in TV and MV in GPR4-sh RNA group but there was no difference in respiratory frequency compared with the Scrambled-shRNA group.The results showed that silencing neuronal GPR4 attenuated the hypercapnic ventilatory response.3.The number of CO₂-sensitive neurons was less in GPR4-shRNA group than that in the Scrambled group.4.Knockdown of GPR4 caused a significant reduction in the pSTAT3protein content（⁵6%）,suggesting that pSTAT3 may be an important component of the GPR4 signal pathway.Summary:GPR4,as a molecular chemoreceptor of NTS neurons,are required for the hypercapnic ventilatory response.Conclusion:A subset of Phox2b-expressing NTS neurons is CO₂-sensitive.Activation of these neurons potentiates central respiratory drive and pulmonary ventilation.Conversely,lesions of these neurons impaires central respiratory chemoreflex.Moreover,neuronal GPR4 is a molecular chemoreceptor involved in the central respiratory chemoreflex.