The Effect Of Glutamate In LH On The Regulation Of Genioglossus Activity

Background and objectiveObstructive sleep apnea(OSA)is one of the most common sleep diseases.It is characterized by partial obstruction or collapse of the upper respiratory tract due to inhibition of genioglossus(GG)function during sleep.Studies have shown that OSA is associated with abnormal regulation of orexin in the brain.Orexin belongs to neuropeptides and is mainly produced and released by orexin neurons.Their soma are mainly in the lateral hypothalamus(LH),perifornical nucleus(PeF)and dorsomedial hypothalamus.DMN),but their fiber project throughout the entire nervous system,and they are closely related to the all intracerebral arousal system and sleep system we have known.Glutamate is the main excitatory neurotransmitter in the brain.The study found that glutamate receptors are expressed on orexin neurons in the LH.Electronmicroscopic immunohistochemistry revealed that glutamate-positive axon terminals make synaptic contacts with dendrites and neuronal cell bodies throughout the lateral hypothalamus.Receptor binding studies have shown that glutamate has certain degree of binding to its receptor in the LH.In addition,the vesicular glutamate transporter 2(VGlut2)immunoreactive axon terminals were shown in the vicinity of orexin neurons,and most of these terminals were found to be in contact with orexin cell bodies or dendrites.Moreover,glutamate and orexin colocalize at the same synaptic terminal but are stored in different vesicles.These two neurotransmitters can be co-released,and orexin neurons can also use glutamate as a neurotransmitter to mediate their signal transduction.Our previous work showed that orexin released from LH can activate the OX1 and OX2 receptors in the hypoglossal motoneuron enhanced GG activity,suggesting that orexin controls opening of the upper airway.Does glutamate of LH involve in the regulation of GG activity? Is this regulatory activity mediated by orexin neurons in LH? Where does the glutamate of LH come from? For now all of these problems have not been clear.Combined with electron microscopy and biotin dextran amine(BDA)anterograde follow-up studies,the LH glutamate may from the lateral parabrachial nucleus(LPB).It is unclear,whether LPB glutamate can regulate the function of orexin neurons in LH.Therefore,this experiment is to investigate whether glutamate in LH involves in the regulation of GG activity.We want to reveal the source of glutamate in LH and the functional synaptic connections between glutamatergic neurons and orexin neurons by using in vitro photogenetic and designer receptors exclusively activated by designer drugs(DREADD).We hope to provide a new theoretical basis for the pathogenesis and clinical treatment of OSA.Materials and MethodsHealthy adult male SD rats,VGlut2-Cre and wild type C57BL/6 mice were used in all experiments.1.SD rats were anesthetized and different concentrations of exogenous glutamate were microinjected in LH and the EMG of ipsilateral GG is recorded.We sought toobserve the effects of different doses of glutamate on GG activity.2.Similarly,to observe effect of endogenous glutamate on GG activity by microinjecting specific glutamate receptor antagonists in LH;3.After SD rats were anesthetized,microinjection was performed in LH,and the ipsilateral GG activity was recorded.Firstly,exogenous glutamate was microinjected and then different specific glutamate receptor antagonists were microinjected.we want to observe the effect of antagonists on exogenous glutamate on GG EMG;4.The optogenetic method combined with in vitro electrophysiological experiments are used to study the functional synaptic connections between glutamatergic neurons in LPB and orexin neurons in LH.After AAV-hSyn-DIO-ChR2-mCherry viruses were injected into the LPB of VGlut2-Cre mice,electrophysiological recordings were performed 3 weeks later to study the projection of mCherry-positive fiber terminals in LH and the reactivity of neurons to 473 nm blue light;5.The DREADD method was used to study the functional expression of the LPB-LH neural circuit.The AAV-hSyn-hM3Dq-mCherry viruses were injected into the LPB of wild type mice,and 3 weeks later,intraperitoneal injection of clozapin-N-oxide(CNO)was performed to study change of the number of c-Fos expressed by neurons in LPB and LH and the c-Fos expression rate of orexin neurons in the LH to clarify the functional expression of the LPB-LH neural circuit.Result1.The effect of exogenous glutamate on GG EMG in rats:Microinjection of different concentrations of glutamate(1 mmol/L,10 mmol/L,100 mmol/L)into the LH of normal rats significantly increased GG activity(P<0.05,n=6),but blood pressure and heart rate of the animals did not significantly change(P>0.05,n=6);2.The effect of specific glutamate receptor antagonists on GG EMG in rats:2.1 Microinjection of specific AMPA glutamate receptor antagonist CNQX(1mmol/L)in the LH of normal rats did not attenuate GG activity(P>0.05,n=6);2.2 Microinjection of specific NMDA glutamate receptor antagonist D-AP5(1mmol/L)in the LH of normal rats reduced GG activity(P<0.01,n=7);3.Specific glutamate receptor antagonists attenuated the effect of exogenous glutamate on GG in normal rats under anesthesia:3.1 Microinjection of glutamate(10 mmol/L)into the LH of normal rats caused an increase in GG activity,and re-injection of the specific AMPA glutamate receptor antagonist CNQX(10 mmol/L)cannot attenuated the enhanced activity of GG by glutamate(P? 0.05,n=5).3.2 Microinjection of glutamate(10 mmol/L)into the LH of normal rats caused an increase in GG activity.After injection of specific NMDA glutamate receptor antagonist D-AP5(10 mmol/L),it could weaken this increase.The activity of GG caused became normal(P<0.05,n=5).4.Functional synaptic connections between glutamatergic neurons in LPB and orexin neurons in LH:A large number of mCherry-positive fibers were observed in the LH under fluorescence microscopy.Excitatory postsynaptic currents(EPSCs)were detected in the LH neurons after exposure them to blue light in patch clamp experiment.This current can be completely blocked by AMPA and NMDA glutamate receptor antagonists,suggesting that there is a functional excitatory synaptic link between LPB and the LH.Further analysis revealed that 45 cells were recorded that 32.4% of the neurons in which glutamatergic neurons in LPB form functional synaptic connections with the LH are orexin neurons.5.To study the functional expression of LPB-LH neural circuit by using DREADD method.Compared with the vehicle control group and the virus control group,the c-Fos expression of the neurons in the LPB and LH increased significantly(P<0.05).In addition,compared with the control group,the c-Fos expression rate of orexin neurons in LH of the experimental group also increased(P<0.05).Conclusion1.Exogenous and endogenous glutamate in LH can enhance the activity of GG;2.Glutamate in LH enhances GG activity mainly by activating NMDA glutamate receptors;3.Glutamatergic neurons in LPB project to orexin neurons in LH;4.Glutamatergic neurons in LPB may play a role in regulating GG EMG by activating orexin neurons in LH.