三叉神经运动核在束缚应激致小鼠咬肌肌电变化中的调控作用

The involvement of trigeminal motor nucleus in the electromyography change of masseter muscle induced by chronic restraint stress in mice

目的观察慢性束缚应激后,小鼠清醒状态下咬肌肌电水平以及支配咬肌运动的三叉神经运动核(trigeminal motor nucleus,Vmo)神经元的变化,为探究心理因素与颞下颌关节紊乱病发生的相关中枢调控机制提供实验依据。方法32只雄性小鼠被随机分为对照组、应激组,对应激组小鼠施加4 h/d、连续14 d的慢性束缚应激;对照组小鼠正常饲养。14 d后,通过旷场实验与高架十字迷宫实验观察小鼠的行为学改变;检测清醒状态下小鼠咬肌肌电水平;采用全细胞膜片钳技术观察Vmo神经元的电生理特性,并利用免疫组织荧光技术观察Vmo内Ⅰ型、Ⅱ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1/2,VGLUT1/2)的表达情况。结果应激组小鼠在旷场实验的中央活动时间(P=0.0004)与中央活动路程(P=0.0004)均显著低于对照组;高架十字迷宫实验中应激组小鼠的开臂进入次数百分比(P=0.0002)与滞留开臂时间百分比(P=0.0013)均显著低于对照组,显示存在明显的焦虑样行为。对照组和应激组小鼠在应激开始前,咬肌累积肌电(integral electromyography,iEMG)(P=0.8779)及振幅均方根(root mean square,RMS)(P>0.9999)均无明显差异;应激结束后,应激组小鼠咬肌的iEMG(P=0.0004)和RMS值(P=0.0001)均显著高于对照组。对照组小鼠在应激前后的iEMG(P=0.7989)和RMS值(P>0.9999)比较无显著差异;应激组小鼠在应激结束后,其咬肌的iEMG(P=0.0011)和RMS值(P=0.0019)显著高于应激前水平。电生理结果显示,在电流钳模式下,当输入60、80、100 pA电流时,应激组小鼠Vmo神经元的放电频率显著高于对照组(P<0.05);应激组小鼠Vmo神经元的自发性兴奋性突触后电流频率(P=0.0030)与幅度(P=0.0002)显著高于对照组。免疫组织荧光染色结果显示,应激组小鼠Vmo部位的VGLUT1(P=0.0010)与VGLUT2荧光强度(P=0.0013)均显著高于对照组。结论慢性束缚应激能够导致小鼠的焦虑样行为及咬肌肌电活动水平的增高。应激后脑内Vmo神经元兴奋性升高,其受到的谷氨酸能兴奋性投射增多,可能是束缚应激造成咬肌肌电活动水平升高的中枢机制之一。

Objective To investigate the changes of the electromyography(EMG)of masseter muscle and excitability of trigeminal motor nucleus(Vmo)neurons induced by chronic restraint stress in mice,aiming to provide an experimental basis for exploring the central mechanism of the relationship between psychological factors and temporomandibular joint disorders(TMD).Methods Thirty-two male mice were randomly divided into control group and stress group.The mice in stress group were subjected to restraint stress for consecutive 14 d(4 h/d).The behavioral changes were evaluated by open-field test and elevated plus maze text.The EMG level of the masseter muscle was measured in the awake state.The electrophysiological properties of the neurons in the Vmo were observed using the whole-cell membrane clamp technique.And the expressions of vesicular glutamate transporter 1/2(VGLUT1/2)in Vmo were assessed by immunofluorescence staining.Results The time spent in center area(P=0.0004)and distance moved(P=0.0004)in center area of mice in the stress group were significantly shorter than those in the control group in the open field experiment.The percentage of open-arm entries(P=0.0002)and the open-arm retention time(P=0.0013)of the mice in the stress group were significantly lower than those in the control group in the elevated plus maze text,indicating a significant anxiety-like behavior.Before the experiment,there was no significant difference in the integral electromyography(iEMG)(P=0.8779)and root mean square(RMS)(P>0.9999)of the masseter muscle between the control and stress group.After the stress process,the iEMG(P=0.0004)and RMS(P=0.0001)of the masseter muscle was higher in the mice of the stress group than in the control group.In the control group,there was no difference in the iEMG(P=0.7989)and RMS(P>0.9999)of the masseter muscle before and after the stress process.In the stress group,the iEMG(P=0.0011)and RMS(P=0.0019)of the masseter muscle at the end of the stress process was significantly higher than the level before it.The electrophysiological results showed that increased spike numbers were elicited in Vmo neurons in stress group compared with control group when currents of 60,80 and 100 pA(P<0.05)were applied in the current clamp mode.The frequency(P=0.0030)and amplitude(P=0.0002)of spontaneous excitatory postsynaptic currents in Vmo neurons in mice of the stress group were significantly higher than that in the control group in the voltage clamp mode.Immunofluorescence staining showed that the fluorescence intensity of VGLUT1(P=0.0010)and VGLUT2(P=0.0013)in the Vmo was significantly higher in the stress group than in the control group.Conclusion Chronic restraint stress can lead to anxiety-like behavior in mice with increased levels of electromyographic activity of masseter muscles.The elevated excitability of Vmo neurons in the brain and the increased glutamergic excitatory projections to them may be one of the central mechanisms leading to the hyperactivity of masseter muscles caused by chronic restraint stress.