Molecular Basis Of Phox2b-Expressing Neuronal Chemosensitivity In Nucleus Tractus Solitarius

Central respiratory chemoreceptors boost an important homeostatic reflex that regulates breathing and CO2 excretion to keep acid-base equilibrium of central and peripheral tissues.Central respiratory chemoreception is completed by brainstem neural circuits composed of anatomically and functionally connected specific neurons or glia sensing changes of CO2/H+ in extracellular fluid surrounding brain tissues.Retrotrapezoid nucleus(RTN)and nucleus tractus solitarius(NTS)are the most important chemoreceptor candidates.Central respiratory chemoreceptor candidates should present at least three requisite properties:(1)inherent sensitivity to physiologically relevant changes in CO2/H+;(2)sensitivity to changes in CO2 that are readily demonstrable in vivo;(3)functional connectivity to respiratory centers,such that their selective activation,inhibition,or elimination produces the appropriate effect on the respiratory chemoreflex.Although NTS neurons basically meet the above demands,it remains to be tested whether NTS chemosensitive neurons have the distinctive neurochemical phenotype and which molecular base underpins such chemosensitivity.Paired-like homeobox 2b(Phox2b)is a homeobox gene coding a homeobox transcription factor specifically expressed in the afferents of respiratory reflex pathways(e.g.carotid body,petrosal and nodose ganglia,NTS,RTN).Phox2b mutations are now considered an important causal for congenital central hypoventilation syndrome(CCHS),as the mutations found in most(>90%)CCHS patients may result in structural and functional deficiencies of central respiratory chemoreceptors.It is evidenced that most Phox2b-expressing RTN neurons sensing extracellular CO2/H+,contributed to regulation of breathing,suggesting that Phox2b is likely a candidate molecular signature for respiratory chemoreceptors.However,it remains unanswered as to whether Phox2b-expressing NTS neurons are intrinsically chemosensitive and whether Phox2b is a specific molecular signature for neuronal chemosensitivity.To address these issues,this project was designed as follows:Objective: We aimed to apply electrophysiological approaches in combination with single cell RT-PCR and examine in Phox2b-EGFP-Jx101 transgenic mice:(1)characterization of Phox2b-expressing NTS neuronal responses to CO2/H+;(2)neurochemical phenotype of chemosensitive NTS neurons.Methods:1 Using PCR to genotype Phox2b-EGFP-Jx101 mice and immunofluorescence staining to reveal whether EGFP-expressing neurons were positively immunoreactive for Phox2b.2 Using cell-attached and whole cell patch clamp configuration to test effects of CO2/H+ changes on spontaneous firing rate,membrane potential and pH-sensitive currents of Phox2b-expressing NTS neurons from acute brain slices.3 Using single cell RT-PCR to analyze the neurochemical phenotypes of Phox2b-expressing chemosensitive neurons.Results:1 Immunofluorescence staining revealed that 100% EGFP-labeled neurons were positively immunoreactive for Phox2b.2 In the presence of pharmacological blockade of synaptic transmissions,a subset of Phox2b-expressing neurons(43%),mostly located in the medial and dorsal parts of NTS,displayed vigorous chemosensitivty.3 Under cell-attached conditions,bath acidification increased spontaneous firing activity in Phox2b-expressing NTS neurons from acute brainstem slices,an effect exhibiting sustained or transient pattern.4 Whole cell current clamp recordings indicated that bath acidification resulted in a membrane depolarization and an increase in firing rate.Whole cell voltage clamp recordings revealed a pH-sensitive current with reverse potential of-85 mV,reminiscent of a background K+ current.5 Single cell RT-PCR manifested that of all the Phox2b-expressing chemosensitive neurons,100% expressed Phox2b and vesicle glutamate transporter 2(VGlut2),as well as 90% expressing G-protein coupled receptor 4(GPR4).Conclusions: We concluded that a subset of Phox2b-expressing NTS neurons displayed intrinsic chemosensitivity,independent of synaptic transmission.The molecular basis of neuronal chemosensitvity was putatively attributed to GPR4 and/or background K+ channels.