Pneumotaxic Modulation On Hering-Breuer Reflex And Hypoxic Respiratory Response

The rostrolateral pontine pneumotaxic center comprised of a dorsal(dl-)and a ventral(vl-)subdivision.The anatomical substrates are KF and parabrachial nuclei for the dl-pons,and A5 for the vl-pons.These two pneumotaxic subdivisions are reciprocally interconnected both functionally and anatomically.The pontine pneumotaxic center participated in respiratory rhythm genesis,respiratory phase-switching,and the coordination of respiratory movements with other somatic activities.The aim of the present study is to decipher the cellular mechanism of pneumotaxic function by analyzing the influences of dl-and vl-pons on two fundamental respiratory reflexes:the pulmonary stretch reflex(also called Hering-Breuer reflex,HBR)as simulated by electrical vagal stimulation,and the peripheral chemoreflex as evoked by a brief hypoxic ventilation.This systemic work comprised of 5 parts.Part-1 and Part-2:influences of certain neurotransmitter antagonists in the dl-and vl-pons on Hering-Breuer reflex(part-1)and on the peripheral chemoreflex or hypoxic respiratory response(part-2).Part-3:Descending axonal projections from dl-pons to brainstem respiratory-related structures as marked by hypoxia-evoked and HBR-evoked c-Fos immunohistology.Part-4:The influences of NMDA receptor antagonist MK-801 on the Hering-Breuer reflex in wild-type or MECP2 knock-out mice.Part-5:Effects of pre-stimulation at vl-pons and MK-801 on the Hering-Breuer reflex.1.Influences of NMDA,GABA and AMPA receptor antagonists in the dl-and vl-pons on Hering-Breuer reflexExperiments were performed on 35 adult SD rats that were anesthetized with urethane,paralyzed with pancuronium,and ventilated artificially.Phrenic nerve discharge was recorded as central respiratory output.Hering-Breuer reflex was simulated with electrical vagal stimulation at the central end with low-intensity (20-80μA),high frequency(80 Hz)and short-pulse duration(0.1 ms)pulses.One episode of stimulation lasted 60 sec.Typically,such electrical vagal stimulation produced immediate phrenic inhibition and the respiration ceased at expiratory phase(Te).This is called HBR stage-1 Te prolongation.The duration of this vagal evoked expiration(stage-1)was stimulation intensity-dependent.However with the continuation of vagal stimulation,rhythmic phrenic discharge reappeared and gradually adapted toward pre-stimulation baseline level.This gradual adaptation of phrenic discharge to vagal stimulation is called the HBR stage-2.At the off-set of the vagal stimulation,the respiratory frequency showed a temporary rebound-like increase,which is called HBR stage-3.After a control HBR was recorded, microinjections of neurotransmitter antagonists were made and another HBR was recorded.The following antagonists were used:NMDA receptor antagonist D-AP5 (10 mM);GABA_A receptor antagonist bicuculline(BIC,5 mM)and AMPA receptor antagonist CNQX(10 mM).The volume of injection was 20-50 nl.Results are as follows:1,the HBR was enhanced following 1)microinjection of D-AP5 into dl-pons,and 2)microinjection of BIC into vl-pons.In these cases the stage-1 Te was significantly longer than control HBR and the adaptation of stage-2 was significantly slower and incomplete.In addition,the shortening of Ti was stronger. The stage-3 rebound was blocked after D-AP5 into dl-pons,but remained unchanged after BIC into vl-pons.2,the HBR was weakened following 1) microinjection of D-AP5 into vl-pons,and 2)microinjection of BIC into dl-pons.In these cases the stage-1 Te was significantly shorter than control HBR and the adaptation of stage-2 was more complete.The shortening of Ti was weaker.The stage-3 rebound was weaker after BIC into dl-pons,but remained unchanged after D-AP5 into vl-pons.3,microinjection of CNQX had no significant influence on HBR.This part of study showed that the pontine NMDA receptor and GABA_A receptor mediated neurotransmissions are involved in the modulation of Hering-Breuer reflex. 2.Influences of NMDA,GABA and AMPA receptor antagonists in the dl-and vl-pons on peripheral chemoreflexThe animal preparations were the same as in part-1.Peripheral chemoreflex was evoked by ventilating the animal with nitrogen-balanced 8%O₂ for 30-50 sec. Under control condition,such hypoxic ventilation caused rapid increase in respiratory frequency and inspiratory amplitude,and shortening of inspiration time. This abrupt increase in respiratory frequency is largely due to the parallel shortening of Te.At the resumption of normal ventilation,the respiratory frequency showed abrupt decline that was mainly due to the parallel prolongation of Te.This abrupt dropping in respiratory frequency is called PHFD(post-hypoxic frequency decline).The peripheral chemoreflex was tested again following microinjections (see part-1 for details concerning the microinjection).Results are as follows:1,the rapid increase in respiratory frequency and the parallel shortening of Te was enhanced after microinjection of BIC in dl-pons.2,the rapid shortening of Te was weakened after microinjection of CNQX into dl-pons,however,the parallel weakening of rapid respiratory frequency increase was not statistically significant, which was due to the enhanced hypoxic shortening of Ti.3,the hypoxic shortening of Ti was enhanced after microinjection of CNQX into vl-pons.4,the PHFD became smaller after microinjection D-AP5 or CNQX into dl-pons,and BIC or CNQX into vl-pons,respectively.5,hypoxic changes in respiratory parameters after microinjections other than those described above were not statistically significant.6, the hypoxic increase in inspiratory amplitude was not affected in any case.This part of study showed that the pontine neurotransmissions mediated by NMDA receptor,GABA_A receptor,and AMPA receptor participated in the modulation of peripheral chemoreflex.In the dl-pons,the NMDA receptor is mainly involved in the PHFD,while the GABA_A receptor mainly in acute hypoxic responses.In the vl-pons,the GABA_A receptor is mainly involved in PHFD and the AMPA receptor is involved in both acute and PHFD.NMDA receptor did not play significant role at vl-pons in modulating peripheral chemoreflex.3.Pontine projections to brainstem structures that are involved in HBR and peripheral chemoreflexExperiments were done on 8 adult SD rats that were anesthetized with pentobarbital.The anterograde tracer biotin dextran(BDA)was injected into dl-pons with pressure(volume 20-30 nl,10%)or electroiontophoresis(positive current,5μA,15-30 min.)under sterile condition.After a post-surgery survival period of 10 days,the animals were either challenged with hypoxia or PEEP (positive end-expiratory pressure)for 2-3 hours.The animals were anesthetized, perfused,post-fixed and the brains were cut into serial sections at thickness of 50μm.Sections were incubated in rabbit anti-cFos primary antibody and biotinated goat anti-rabbit IgG.The c-Fos and BDA labeling were visualized using avidin-biotin-HRP histochemistry with DAB as chromogen.It was found that c-Fos immunopositive neurons mainly localized in NTS(medial,commissural, ventrolateral)and ventrolateral medulla following either hypoxia or PEEP. Anterogradely labeled axonal terminals from dl-pons were found to reach well-established brainstem respiratory related structures and overlap with c-Fos immunopositive neurons in NTS and ventrolateral medulla in and around the ambiguus nucleus,where the ventral group of respiratory neurons were recorded.In addition,single axon from KF nucleus sent out branches to innervate multiple brainstem structures.This study revealed anatomical connections between dl-pons and HBR or peripheral chemoreflex related neurons,underscoring our findings in part-1 and part-2 of the present research.4.Hering-Breuer reflex in wild-type and MECP2 knock-out miceRett syndrome is a severe neurodegenerative disease that afflicts tens of thousand children.One of the most prominent symptoms of Rett syndrome is the apneusis or inspiratory withholding.About 26%Rett syndrome patients died of respiratory failure.It was revealed that Rett syndrome is caused by mutation of MECP2 gene that encodes methyl-CpG combining protein.We hypothesize that the respiratory abnormality is caused by the malfunction of Hering-Breuer reflex.This part of research was performed on wild type(+/+)and heterozygous MECP2 knock-out(+/-)mice.Mice were anesthetized with urethane,paralyzed,and artificially ventilated.Phrenic nerve discharge was recorded as central respiratory output.Hering-Breuer reflex was simulated with sustained electrical stimulation at the central cut-end of cervical vagus nerve.Stimulation parameters were the same as in part-1.The results are as follows:1,in +/+ mice,sustained vagal stimulation produced similar stage-1 HBR as in rats as described in part-1.However,the stage-2 adaptation led to net increase in respiratory frequency higher than pre-stimulation value.We call this net increase in respiratory frequency in stage-2 "delayed frequency increase",which is largely due to the progressive shortening of Te in stage-2.Stage-3 rebound was much weaker than in rats thus not analyzed in this part of study.2,in 8 +/-mice,the stage-1 HBR was similar to that of +/+ mice. However,4 of them failed to exhibit stage-2 "delayed frequency increase".The other 4 exhibited "delayed frequency increase" as in +/+ mice.3,Following systemic application of the NMDA receptor antagonist MK-801,the stage-2 "delayed frequency increase" was not observed in either +/+ or +/-mice.This part of study suggested that the HBR was defective in at least part of MECP2 knock-out mice,which might contribute to the respiratory failure observed in patients suffering from Rett syndrome.5.Pre-electrical stimulation and microinjection of MK-801 at vl-pons modulated the Hering-Breuer reflexExperiments were performed on 38 adult Wistar rats under urethane anesthetization,gallamine triethiodide paralysis and artificial ventilation.Phrenic nerve discharge was recorded as central respiratory output.HBR was simulated with electrical vagal stimulation at the following parameters:intensity 20-40μA (1.5 threshold),frequency 80 Hz,pulse duration 0.1 ms,stimulation time 60 sec. Parameters for vl-pons stimulation:intensity 50-80μA,frequency 80 Hz,pulse duration 0.3 ms,stimulation time 20 sec.Immediately following vl-pons stimulation, another vagal stimulation caused stronger HBR than control,with stage-1 Te increased,adaptation weakened,and stage-3 rebound weakened too.Following microinjection of MK-801 at the vl-pons,another vagal stimulation caused weaker HBR than control,with stage-1 Te shortened,adaptation more complete,and stage-3 rebound weakened.This part of study supplied another proof that the vl-pons modulates the Hering-Breuer reflex.This part of research has been published in Adv Exp Med Biol,vol.605,2008).