The Neural Pathway Mechanism Of Anxiety-like Behavior Induced By Abnormal Occlusion

Temporomandibular joint disorder(TMD)is a common disease in the oral and maxillofacial region,which is often manifested as orofacial pain,joint bounce and mandibular movement limitation.Abnormal occlusion is a common cause of temporomandibular joint disorder.Its main neural mechanism is that abnormal occlusion can cause the excitation of periodontal proprioceptors,activate the trigeminal mesencephalic nucleus(Vme),activate the motor neurons through synaptic connection,and lead to the abnormal contraction of skeletal muscles such as masticatory muscles,and then affect the normal movement of joints and the stress of condylar cartilage,eventually leads to temporomandibular joint osteoarthritis.Abnormal occlusion can cause the excitation of neurons in the Vme and the dorsomedial part of the principal sensory trigeminal nucleus(Vpdm).Vpdm and the ventral posteromedial nucleus(VPM)of the thalamus have been proved to be the key relay stations for proprioception transmission.VPM send a large number of neural projections to the primary sensory area(SI),which regulates emotion.Many TMD patients suffer from negative emotions such as anxiety and depression.Whether the trigeminal proprioception pathway is involved in the regulation of anxiety-like emotion caused by abnormal occlusion is lack of detailed research.Abnormal occlusion can be treated as a chronic stress to cause metabolic disorder of the hypothalamic-pituitary-adrenal axis(HPA axis).The abnormal metabolism of HPA axis often causes negative emotions such as anxiety.The neuroendocrine activity of HPA axis is mainly regulated by neurons that release corticotropin releasing hormone(CRH)in the paraventricular nucleus(PVN)of the hypothalamus.PVN receives direct projections from multiple neural nuclei in different regions of the brain.There is a gap connection between the neurons of Vme and the neurons of the most important noradrenergic nucleus,locus ceruleus(LC).LC is confirmed to have projection to PVN.However,it is not clear whether abnormal occlusion will cause the excitation of LC and PVN neurons,and what role the LC to PVN neural pathway plays in the HPA axis metabolic disorder and anxiety-like mood caused by abnormal occlusion.Recently,we have developed an abnormal dental occlusion model termed unilateral anterior crossbite(UAC)model that could induce serious osteoarthritic degeneration of temporomandibular joint cartilage.UAC can lead to abnormal excitation of skeletal muscles in the oromaxillofacial region of rats,such as masticatory muscles;At the same time,it can also lead to anxiety.Based on the UAC animal model,this experiment comprehensively illustrated the role of Vpdm-VPM proprioceptive neural pathway and LC-PVN neural pathway in HPA axis excitability abnormality and anxiety-like emotion caused by abnormal occlusion by using a variety of morphological,neural tract tracking,electrophysiological,chemical lesion,chemical genetics,optogenetics and behavioral pharmacological approaches.Therefore,this study is mainly divided into the following two sections:Part One:Mechanism of Vpdm-VPM neural pathway in the regulation of anxiety-like behavior induced by UACObjective:To explore the effect of abnormal occlusion on trigeminal proprioceptive neural pathway and the effect of specific regulation of Vpdm-VPM neural pathway on UAC-induced anxiety behavior.Methods:(1)The effects of specific modulation of VGLUT1 positive neurons in Vpdm on anxiety-like behavior induced by UAC were observed by using chemogenetics,immunofluorescence and behavioral methods;(2)BDA anterograde tracing combined with fluorogold(FG)retrograde tracing was used to verify the existence of the neural pathway of Vme-Vpdm-VPM-SI for the proprioception upward transmission;(3)The activation of VGLUT1 neurons in Vpdm and VGLUT1 positive neurons in Vpdm to VPM of UAC model were detected by the combination of FG retrograde tracing,in situ and fos immunofluorescence;(4)The influence of Vpdm-VPM neural pathway on anxiety-like behavior were detected by means of chemogenetics;(5)optogenetic methods were used to inhibit the projection of VGLUT1 neurons in Vpdm to VPM and observe the effect on anxiety-like behavior.Results:(1)The specific inhibition of VGLUT1 positive neurons in Vpdm of VGLUT1-Cre mice by chemogenetics alleviated the anxiety-like behavior caused by UAC.Specific activation of neurons lead to anxiety-like behavior in normal animals,but failed to aggravate the negative emotional behavior of UAC animals.(2)There exists a neural pathway of Vme-Vpdm-VPM-SI for upward proprioception transmission,which can transmit orofacial proprioception information from the trigeminal nucleus to the primary sensory cortex.(3)UAC caused the activation of VGLUT1 neurons in Vpdm and VGLUT1 positive neurons in Vpdm to VPM.(4)Chemogenetic inhibition of Vpdm-VPM neural pathway relieved the anxiety-like behavior of UAC mice;The specific activation of chemical genetics lead to anxiety in normal animals,but did not worsen the negative emotions of UAC animals.(5)Optogenetic inhibition of the projection of VGLUT1 positive neurons in Vpdm to VPM of VGLUT1-Cre mice alleviated the anxiety-like behavior induced by UAC,but did not affect the emotional state of normal mice.Conclusion:There exists VmeVGLUT1-VpdmVGLUT1-VPM-SI,a trigeminal nerve-thalamic-cortical neural pathway,which transmits proprioception information to the cortex.UAC can cause abnormal excitation of VGLUT1 positive neurons projecting to VPM in Vpdm.Vpdm-VPM neural pathway is involved in the regulation of anxiety-like behavior in UAC mice.Specific inhibition of Vpdm-VPM projection pathway reduced the anxiety-like performance caused by UAC,and the activation can lead to the generation of anxiety-like negative emotion in animals,and this process is mainly mediated by VGLUT1.Part Two:The mechanism of LC-PVN neural pathway in the UAC induced HPA axis disorder and anxiety-like behaviorObjective:To demonstrate UAC can lead to HPA axis metabolic disorder and anxiety-like behavior,and explore the mechanism of LC-PVN neural pathwayMethods:(1)BDA anterograde tracing,ELISA detection,real-time quantitative PCR and electrophysiological research methods were used to observe the neural projection of LC to PVN,and the expression of corticosterone,the expression of TH mRNA in LC and CRH mRNA in PVN,and the excitability changes of LC and PVN neurons were measured in UAC model;(2)The effects of damaged PVN nuclei on the expression of serum CORT and anxiety-like behavior of animals were observed by the methods of using chemical lesions of kainic acid(KA),ELISA,immunofluorescence,open field test and elevated plus maze test;(3)The effects of chemical genetics on the expression of CORT and anxiety-like behavior were observed by specifically activating or inhibiting CRH neurons in PVN;(4)The neural projection of LC to CRH neurons in PVN was checked by using rabies virus trans-single synaptic labeling system;(5)The LC-PVN neural pathway was specifically regulated by chemical genetics,and the effect on CORT expression and anxiety-like behavior was observed;(6)Optogenetic method was used to regulate the projection of TH neurons in LC to PVN,and pharmacological method was used to locally administer α1 and β adrenoceptors in PVNto observe the effect of specific antagonist on anxiety-like behavior.Results:(1)The BDA-labeled axon terminals were observed around PVN neurons after the anterograde tracer biotinylated dextran amine(BDA)was injected into LC;UAC resulted in increased expression of serum CORT,TH mRNA in LC and CRH mRNA in PVN,and increased excitability of neurons in LC and PVN.(2)The activity of CRH-positive neurons in the PVN of CRH-Cre mice was specifically inhibited by chemical lesion and chemogenetic approach.It was observed that the CORT expression of UAC was reduced,and the anxiety-like behavior caused by UAC was relieved.The specific activation of CRH positive neurons in PVN by chemical genetics increased the expression of CORT,induced anxiety in normal animals,and aggravated the negative emotion of UAC animals.(3)The results of rabies virus trans-single synaptic labeling and immunofluorescence suggest that after injecting RV virus into the PVN of CRH-Cre mice,the GFP transsynaptic labeled neurons projecting to CRH-positive neurons in PVN were observed in LC,and most of them were TH-positive neurons.(4)Specific inhibition of LC-PVN neural pathway by chemogenetic approach reduced the expression of CORT in UAC mice and relieved the anxiety-like behavior of UAC mice;However,the specific activation of LC-PVN neural pathway by chemogenetics increased the expression of CORT and lead to anxiety-like behavior in normal animals,but failed to further worsen the anxiety-like behavior of UAC animals.(5)Optogenetics that specifically inhibits the projection of TH positive neurons in LC to PVN alleviated the anxiety-like behavior caused by UAC;Optogenetics combined with behavioral pharmacology indicated prazosin,the epinephrine α1 receptor antagonist,can alleviate the anxiety-like behavior of animals caused by optogenetic stimulation;Antagonism with local application of propranolol β-AR did not alleviate the anxiety-like behavior of animals caused by optogenetic stimulation.Conclusion:UAC can induce the excitation of LC and PVN neurons,thus causing the metabolic disorder of HPA axis and the increase of CORT expression,leading to anxietylike behavior in animals.Specific inhibition or activation of CRH neurons in PVN can regulate HPA axis disorder and anxiety-like behavior caused by UAC.TH-positive neurons in LC can project to PVN and form synaptic contact with CRH-positive neurons in PVN.The specific inhibition or activation of LC-PVN neural pathway can cause changes in the expression of serum CORT and regulate the negative emotion caused by UAC.The activation of the projection pathway from TH positive neurons in LC to PVN is mainly regulated by α1-adrenergic receptor in PVN.