Modulation And Mechanism Of Genioglossus By 2-APB At The Hypoglossal Motor Nucleus In Rats

Objective: Obstructive sleep apnea(OSA)is a common sleep breathing disorder.The first line treatment for OSA is non-invasive positive pressure ventilation,however,poor patient compliance may limit the effectiveness of treatment.Finding effective drug therapeutic targets may be crucial for the treatment of this disease.OSA is characterized by recurrent upper airway collapse during sleep,which indicates that the decreased muscle tone of upper airway dilators associated with sleep plays a key role in the pathogenesis of OSA.To study the regulation of genioglossus and find suitable drug therapeutic targets is an effective way to study drug therapy for this disease.The change of intracellular calcium([Ca2+]i)concentration is a universal signal mechanism,which is often accompanied by the change of membrane potential and participates in the regulation of neuronal excitability.Hypoglossal motor neurons(HMs)directly regulate genioglossus activity and are affected by a variety of neurotransmitter and ion channels.Many regulatory mechanisms can increase genioglossus activity by regulating calcium ions in HMs,such as histamine and 5-HT.2-aminoethoxydiphenyl borate(2-APB)is a common [Ca2+]i regulator in physiological research.The regulattion are various.Evidences showed that it can increase cell excitability through [Ca2+]i elevation.The role and mechanism of 2-APB in regulating genioglossus activity through HMN is still unclear.The purpose of this study is to explore the role of 2-APB in regulating genioglossus activity through HMs and its related ion channels and signal transduction mechanisms in vivo and in vitro so as to further improve the central regulatory mechanism of OSA,and find relevant drug therapeutic targets,providing a theoretical basis for the drug treatment of OSA.Methods:(1)Effects of different concentrations of 2-APB on regulation of genioglossus activity at HMN After the rats were anesthetized with isoflurane,an indwelling cannula was placed in the HMN on one side of medulla oblongata by brain stereolocalization technology.At the same time,electrodes were implanted to record the electromyogram of genioglossus,diaphragm and neck muscle,and tension transducer was used to record the respiratory rate of rats.The rats recovered for half an hour after operation.When all indexes were stable,they were injected with artificial cerebrospinal fluid(ACSF)for 300 nl and recorded the above indexes for half an hour as the baseline.Then,they were given 2-APB of different concentrations(5m M,10 m M,20 m M,300nl/time)at the same injection site.Each concentration was recorded for half an hour,and the next concentration was continued after the genioglossus electromyogram returned to baseline.Study the effects of 2-APB at different concentrations on genioglossus electromyogram(including tonic and respiratory-related),diaphragm electromyogram,respiratory rate and neck muscle electromyogram.(2)Effects of HMN administration of different types of ion channel blockers on 2-APB excitation of genioglossus activity under anesthesia Sodium-calcium exchanger(NCX)inhibitors benzamil(5m M),nickel chloride(20m M);intracellular calcium regulator cyclopiazonic acid(CPA)(5m M),BAPTAAM(1,2-bis(2-aminophenoxy)ethane-n,N,n ’-tetraacetic acid)(0.2m M),ruthenium red(5m M);nonselective cation channel blocker lanthanum chloride(20m M)L-type calcium channel(LTCC)blockers verapamil(10m M),diltiazem(10m M)and sodium channel blocker procaine(20m M)were injected into rat HMN in advance or at the same time with 2-ABP(10m M).Compare the effects of the above drugs on the excitation of genioglossus activity by 2-APB.(3)Whole-cell patch clamp was used to further verify the excitatory effect of 2-APB on HMs Acute brain slices were prepared from medulla oblongata HMN of rats about 8 weeks.The effects of 1m M 2-APB on AP firing rate and membrane potential of HMs were recorded and analyzed by current clamp technique.The effect of 2-APB on the AP firing rate and membrane potential of HMs after calcium-free ACSF and 30 min incubation with 0.5m M CPA in advance was subsequently observed.Results:(1)Effects of different concentrations of 2-APB on regulation of genioglossus activity at HMN: Injection of 2-APB with different concentrations(5m M,10 m M,20 m M,300nl/time)in HMN increased genioglossus activity,which was concentration dependent,and the duration of increased genioglossus activity was longer at high concentration.The difference of genioglossus activity between 10 m M and 20 m M was statistically significant compared with ACSF.The tonic related genioglossus activity increased more significant and the duration were longer than respiratory-related genioglossus activity.The 2-APB increased the tonic related genioglossus activity by 824.07%,and increased the respiratory related genioglossus activity by 140.06% at 20 m M.But it had no effect on respiratory rate,diaphragm electromyogram and neck muscle electromyogram.(2)Ion channels associated with the regulation of genioglossus by 2-APB at HMN 1)Pretreatment the NCX inhibitors benzamil and nickel chloride can reduce the excitatory effect of 2-APB on genioglossus activity,among which benzamil can significantly reduce genioglossus activity compared with ACSF.2)Pretreatment the intracellular calcium regulators CPA,BAPTA-AM and ruthenium red can block the excitatory effect of 2-APB on genioglossus activity.Ruthenium red,in addition to inhibiting the excitability of 2-APB,further reduced the activity of genioglossus electromyogram compared with the baseline.3)Pretreatment the LTCC blockers verapamil and diltiazem,but not the nonselective cation channel blocker lanthanum chloride,can block the excitatory effect of 2-APB on genioglossus,of which verapamil reduces genioglossus electromyogram more obviously,which is statistically different compared with ACSF.4)Procaine did not completely block the increase of genioglossus activity induced by 2-APB,and the difference was statistically significant compared with ACSF.(3)Excitatory effects of 2-APB on HMs in vitro In whole-cell patch-clamp experiment,1m M 2-APB could increase the AP firing rate and membrane potential of HMs,and the difference was statistically significant compared with ACSF.Calcium-free ACSF had no significant effect on the increase of AP firing rate and membrane potential induced by 2-APB in HMs,and were statistically significant between calcium-free ACSF and 2-APB.Pretreatment with CPA could block the increase of AP firing rate and membrane potential induced by 2-APB in HMs,and there was no significant difference compared with that before 2-APB administration.Conclusions(1)2-APB can increase genioglossus activity in a concentration-dependent manner,and the tonic-related genioglossus electromyogram increased more obvious and lasted longer.(2)The increase of genioglossus activity induced by 2-APB was related to the increase of intracellular calcium concentration in HMs,which may activate NCX and cause cell depolarization by promoting the release of endoplasmic reticulum calcium storage.LTCC was involved in the regulation of genioglossus activity by 2-APB.