Mechanism Underlying Glutamatergic Neurons In The Posterior PPT Of Rat Enhancing The Genioglossus Muscle Activity

BackgroundObstructive sleep apnea(OSA)is a sleep-related respiratory disease with high incidence in the population.Its main characteristics is repeated obstruction of the upper airway during sleep which leads to the reduction or stop of ventilation,hypertension,myocardial infarction and other cardiovascular and cerebrovascular diseases,traffic and industrial accidents,and even sudden death.The genioglossus muscle(GG)is the largest upper airway dilator in the pharynx.It maintains the upper airway unobstructive.It is dominated by the hypoglossal motoneuron(HMN)within the hypoglossal nucleus(HN).Studies have shown that the pathogenesis of OSA is related to the decreased muscle tone of GG during sleep.In OSA patients,GG activity is relatively strong to keep the airway open during wakefulness.While sleep onset,especially during rapid eye movement(REM)sleep,GG tension is significantly inhibited,leading to severe upper airway obstruction in patients with narrowed anatomical structure of airway.The pedunculopontine tegmental nucleus(PPT)is a part of the reticular formation of brain stem which is involved in a variety of physiological functions.PPT consists of three different types of neurons: glutamatergic,γ-aminobutyrate(GABAnergic)and cholinergic neurons.Studies have found that the activation of glutamatergic neurons in PPT can induce long-term cortical activation and behavioral arousal.Injection of glutamate into PPT can increase wakefulness and REM sleep.So glutamate in PPT is closely related to sleep.Microinjection of glutamate into medio-posterior part of the PPT also enhanced GG EMG.Glutamate(Glu)is the main excitatory neurotransmitter in the central nervous system,which is involved in a variety of physiological functions of the body,such as sleep and wakefulness,cognition,motor control,and so on.There are three main types of ionotropic glutamate(i Glu Rs)receptors: the N-methyl-D-aspartate(NMDA)receptor,the kainic acid(KA)receptor,and the α-amino-3-hydroxy-5-methyl-4-isoxazole(AMPA)receptor.It was found that microinjection of two i Glu Rs NMDA and AMPA antagonist,could inhibit the GG activity.These studies indicate that glutamate is related to GG activity to some extent.Glutamate in PPT can regulate GG activity.So how does glutamate in PPT regulate GG activity? Which glutamatergic receptor is responsible for this regulation?Is this effect mediated by glutamatergic neurons? Is there a direct fiber projection relationship between PPT nucleus and HMN? In order to explain these problems,this study intends to investigate the mechanism underlying glutamatergic neurons in PPT participating in the regulation of GG activity by neurophysiological and neuropharmacological techniques.We hope to provide a new theoretical basis for the pathogenesis and clinical treatment of OSA.Materials and MethodsHealthy adult male SD rats,weighing 250-350 g,were fed a free diet.Animals are anesthetized with 20% uratan or 6% chloral hydrate.1.The EMG activity of ipsilateral GG,mean arterial blood pressure,heart rate and respiratory rate of the were recorded after glutamate was microinjected into theposterior PPT of anesthetized rats.The effects of glutamate with different concentration on GG EMG,mean arterial blood pressure,heart rate and respiratory rate of the rate were observed.2.The EMG activity of ipsilateral GG,mean arterial blood pressure,heart rate and respiratory rate of the were recorded after the NMDA receptor-specific antagonist D-AP5 and AMPA receptor-specific antagonist CNQX was microinjected into theposterior PPT of anesthetized rats.The effects of glutamate with different concentration on GG EMG,mean arterial blood pressure,heart rate and respiratory rate of the rate were observed.3.Observe the effects of the antagonists on the changes of GG EMG induced by exogenous glutamate after two specific glutamatergic receptor antagonists and glutamate were microinjected into the posterior PPT of rats.4.Observe projection of nerve fiber betweentheposterior PPT and the HN nucleus after biotinylated dextran amines(BDA)and choleratoxin subunit B(CTB)were separately injected into the posterior PPT of rats.5.Observe the c-Fos expression rate of glutamatergic neurons in theposterior PPT after microinjecting glutamate in the posterior PPT of rats.Results1.Glutamate microinjection in the posterior PPT enhanced GG EMG in rats:GG EMG was significantly enhanced after glutamate microinjection in the posterior PPT(0.01 nmol,0.1 nmol)in rats(P < 0.05,n=5),but the average arterial pressure,heart rate and respiratory rate of the animals were not affected;2.Microinjection of glutamate receptor specific antagonist in he posterior PPT inhibited GG EMG in rats:2.1 GG EMG was significantly weakened after microinjection of different dose of NMDA receptor-specific antagonist D-AP5(0.01 nmol,0.1 nmol)in the posterior PPT(P < 0.001,n=6),but the mean arterial pressure,heart rate and respiratory rate were not changed;2.2 GG EMG was significantly weakened after microinjection of different dose of AMPA receptor-specific antagonist CNQX(0.01 nmol,0.1 nmol)in the posterior PPT(P < 0.01,n=6),but the average arterial pressure,heart rate and respiratory rate were not changed.3.Microinjection of glutamate receptor-specific antagonist in the posterior PPT decreased the enhancement effect of exogenous glutamate on GG EMG in rats:3.1 Microinjection of NMDA receptor antagonist D-AP5(0.1 nmol)into the posterior PPT of rats attenuated the enhancement effect of GG EMG induced by glutamate(0.1 nmol)(P < 0.001,n=5).3.2 Microinjection of AMPA receptor antagonist CNQX(0.1 nmol)into the posterior PPT of rats attenuated the enhancement effect of GG EMG induced by glutamate(0.1 nmol)(P < 0.001,n=6).4.Projection Nerve fiber between the posterior PPT and HN:After the unilateral microinjection of BDA in the posterior PPT,a large number of neurons in the posterior PPT and nerve fibers in the HN of medullawere labeled anterogradely by BDA.A large number of nerve fibers were labeled in the HN after the unilateral microinjection of CTB in the HN of medulla.Neurons retrogradely labeled by CTB were observed in the posterior PPT,indicating that the neurons in the PPT projects axons directly to the HMN in the HN.5.Glutamate can activate glutamatergic neurons in the posterior PPT:An increase in c-Fos expression was observed in the posterior PPT 1 hour after microinjection of glutamate in the posterior PPT.Conclusion1.Both exogenous and endogenous glutamate in the posterior PPT of rats can enhance the GG EMG by activating NMDA and AMPA glutamate receptors.2.Glutamatergic neurons in the posterior PPT of rats may play an important role in controlling GG activities.3.The neurons in the posterior PPT of rats can directly project to the HN in the medulla and participate in the regulation of HMN on GG.