Effect Of GABA In LH On The Regulation Of Genioglossus Muscle Activity In Rats

BackgroundObstructive sleep apnea（OSA）is a common condition characterized by a normal breathing pattern during wakefulness but repetitive episodes of upper airway part or total obstruction during sleep.It can cause hypoxia and carbon dioxide retention in the body,which can lead to snoring at night,headache when patients wake up,daytime sleepiness which can lead to work-related injuries and traffic accidents,cognitive decline,high blood pressure,heart failure and even sudden death.The genioglossus（GG）is the main dilator muscle of the upper airway and one of the most widely studied muscles.Its movement is controlled by the medulla hypoglossal motoneuron（HMN）.During inspiration its activity increases,making the upper airway dilate.During expiration the muscle activity decreases,making the upper airway narrow.During sleep,GG plays a very important role in the patency of the upper airway.The enhancement of GG activity has important significance in preventing the hypotonia of upper airway during sleep.It has been found that orexin participates in the regulation of GG activities,and orexin content changes in OSA patient’s brain.Orexin is a neuropeptide,secreted by orexinergic neurons.These somas are mainly distributed in the lateral hypothalamus（LH）,perifornical nucleus（Pe F）and dorsomedial hypothalamus（DMN）,but their nerve fibers project to the entire central nervous system except the cerebellum,such as HMNs in medulla,which express orexin receptors.Orexin has a variety of biological functions such as regulating sleep,energy metabolism,breathing and cardiovascular activities,and so on.γ-Aminobutyrate（GABA）is an important inhibitory neurotransmitter in the brain.Up to a third of the brain’s central neurons use GABA as their main neurotransmitter.It plays an important role in sleep/wake,feeding,treatment of mental illness and hormone regulation.GABA receptors consist of three subtypes:GABA_A,GABA_B and GABA_C receptors.GABA_A and GABA_C receptors are ligand-gated chloride ion channels receptors,and GABA_B receptors are G-protein-coupled receptors.LH contains a large number of GABAergic inter neurons and receives GABAergic input from other regions,such as the GABAergic neurons in the preoptic nucleus（POA）region that promote sleep.GABA_A and GABA_B receptors are found in various neurons in LH,including orexinergic neurons and melanin concentration hormone（MCH）neurons.In vitro studies have shown that GABA inhibits the function of these neurons.Our previous studies have demonstrated that orexin released by LH can activate OX₁ and OX₂ receptors in HMNs of medulla,and enhance GG activity,which suggest that orexin is involved in controlling the opening of the upper airway.Is GABA in LH involved in regulating GG activity?Is the regulation mediated by orexinergic neurons in LH?Therefore,the purpose of this study is to explore whether GABA in LH is involved in orexinergic neurons’regulation of GG activity.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 g-350 g were used in all experiments.Food and water were given ad libitum.Rats were anesthetized with urethane or chloral hydrate.1.Observe the neuronal projections between LH and hypoglossal nucleus（HN）of medulla by using anterograde tracer biotinylated dextran amines（BDA）and retrograde tracer cholera toxin subunit B（CTB）.2.The EMG activity of ipsilateral GG,mean arterial blood pressure,heart rate and respiratory rate of the rats were recorded after the GABA_A receptor antagonist gabazine was microinjected into the LH of anaesthetized SD rats.The effects of gabazine with different concentrations on GG EMG,mean arterial blood pressure,heart rate and respiratory rate of the rats were observed.3.The EMG activity of ipsilateral GG,mean arterial blood pressure,heart rate and respiratory rate of the rats were recorded after the GABA_B receptor antagonist CGP55845 was microinjected into the LH of anaesthetized SD rats.The effects of CGP55845 with different concentrations on GG EMG,mean arterial blood pressure,heart rate and respiratory rate of the rats were observed.4.The EMG activity of ipsilateral GG of the rats were recorded after the GABA_Areceptor antagonist muscimol or the antagonist receptor of GABA_B baclofen was microinjected into the LH of anaesthetized SD rats.The effects of muscimol or baclofen on GG EMG of the rats were observed.5.Observe the c-Fos expression rate of orexin neurons in LH after microinjection of GABA_A receptor antagonist gabazine in LH of SD rats.Results1.Projection of nerve fibers between LH and HN of medulla:After unilateral microinjection of BDA into LH,a large number of neurons in LH were labeled by BDA and anterograde BDA labeled nerve fibers were observed in the medullary HN.After unilateral microinjection of CTB into HN of medulla,a large number of nerve fibers were labeled in HN,and neurons labeled retrograde by CTB were observed in LH,indicating a direct axonal connection between the two nucleuses.2.Effect of GABA_A receptor-specific antagonists on GG EMG in rats:Microinjection of GABA_A receptor antagonists gabazine with different concentrations（3 mmol/L,30 mmol/L）in LH of normal rats significantly enhanced GG activity（P<0.01,n=6）;meanwhile,the mean arterial blood pressure of rats was increased（P<0.01,n=6）,heart rate（P<0.01,n=6）,and respiratory rate was also increased.（P<0.05,n=6）;3.Effect of GABA_B receptor-specific antagonists on GG EMG in rats:Microinjection of GABA_B receptor antagonist CGP55845（1 mmol/L,10 mmol/L）in LH of normal rats significantly enhanced GG activity（P<0.05,n=6）;meanwhile,the mean arterial blood pressure in rats was increased（P<0.01,n=6）,heart rate（P<0.01,n=6）,and respiratory rate was increased（P<0.05,n=6）;4.Effect of GABA receptor-specific agonists on GG EMG in rats:Microinjection of GABA_A receptor agonist muscimol（1 mmol/L）in LH of normal rats significantly decreased GG activity（P<0.01,n=6）,and GABA_B receptor-specific agonist baclofen（5 mmol/L）in LH of normal rats significantly decreased GG activity（P<0.01,n=5）;5.Effect of GABA_A receptor antagonists in LH on orexinergic neurons:Microinjection of GABA_A receptor antagonist gabazine in LH,the number of c-Fos expressed in neurons in LH was observed to increase significantly 1 hour later（P<0.001,n=6）.In addition,gabazine can significantly increase the c-Fos expression rate of orexin neurons in this area（P<0.001,n=6）.Conclusion1.Endogenous GABA in LH can inhibit GG activity which is mediated by GABA_A and GABA_B receptor in rats.2.GABA in LH may regulate GG activity by inhibiting orexinergic neurons of the same region in rats.