关键词: 束缚应激, 焦虑样行为, 咬肌肌电, 三叉神经运动核, 全细胞膜片钳, 囊泡膜谷氨酸转运体

Abstract:

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=0.000 4) and distance moved (P=0.000 4) 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=0.000 2) and the open-arm retention time (P=0.001 3) 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=0.877 9) and root mean square (RMS) (P>0.999 9) of the masseter muscle between the control and stress group. After the stress process, the iEMG (P=0.000 4) and RMS (P=0.000 1) 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=0.798 9) and RMS (P>0.999 9) of the masseter muscle before and after the stress process. In the stress group, the iEMG (P=0.001 1) and RMS (P=0.001 9) 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=0.003 0) and amplitude (P=0.000 2) 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=0.001 0) and VGLUT2 (P=0.001 3) 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.

Key words: restraint stress, anxiety-like behavior, masseter muscle electromyography, trigeminal motor nucleus, whole-cell membrane clamp, vesicular glutamate transporter