The Effects Of Psychological Stress On Masseter Muscle Spindle And Muscle Spindle Afferent In Rats, And The Mechanisms Underlying It

With the rapid development of modern society, increasingly intense social competition, and fast-paced living situation, more and more people are nowadays undertaking various physical or psychological stress. More and more systematic diseases have been proved to be related with stress and high pressure. As the main components of stomatognathic system, jaw muscles undertake the important functions including maintaining jaw stable, masticatory movement, jaw growth and so on. A plenty of researches have proved that abnormal psychological situation is highly related with abnormality of structure and function of jaw muscles, including muscle fatigue, muscle hyperalgesia and ultrastructure changes. The previous studies of our research group have demonstrated that psychological stress could activate the related neural nucleuses innervating jaw movement, enhance the transmission of glutamate(one kind of main excitatory transmitters in central neural system), cause the masseter overactivity, and directly or indirectly resulting in pathological changes of masseter muscle. Jaw muscles, especially the deep masseters, contain a lot of proprioceptors—muscle spindles. The main function of muscle spindle is to receive the traction, stretch or the velocities of muscle contraction, and transmit proprioceptive information into the central neural system. Studies have indicated that masseter muscle spindle afferent have complex neural connections with neural nucleuses in the brain innervating jaw movement and is known to play a significant role in oral-motor circuits. Masseter muscle spindle afferent belongs to be pseudounipolar neurons whose somata locate in the Mesencephalic trigeminal nucleus(Vme). Furthermore, the Vme neurons are unique among the primary sensory neurons in that their cell bodies reside in the brainstem. Therefore, the excitability of the Vme neurons are not only affected by the jaw muscle function, but also regulated by the synaptic contacts received from the other nucleus of the brain. It has been demonstrated that Vme neurons are glutamatergic neurons, and the excitatory transmitter glutamate and its receptors play an important role in the jaw muscle proprioception transmission by muscle spindle afferent. So, what’s the effects of psychological stress on masseter muscle spindle and muscle spindle afferent, and whether the glutamate and its receptors are involved in the process, still remain unknown.In the present study, by establishing the animal model of restraint stress in rats, using the behavioral tests, ELISA tests, histopathology, whole-cell patch clamp, immunohistofluorescence and transmission electron microscope examining, we explored the effects of psychological stress on masseter muscle spindle and muscle spindle afferent. In addition, we investigated the involvement of glutamate transmission and N-methyl-D-aspartate receptors(NMDARs) underlying the process by using the NMDARs antagonist MK801.There two parts of experiments in the present study:Part 1: The effects of psychological stress on masseter muscle spindle and muscle spindle afferent in rats.Experiment 1: The establishment and evaluation of psychological stress animal models.SD Rats were randomly distributed into four groups: control group, stress group, Low dose MK801 group(0.1mg/kg) and high dose MK801 group(0.2mg/kg). Psychological stress was induced by subjecting rats to restraint stress(6 h/d) for 14 consecutive days. The MK801 were administrated by intraperitoneal injection every other day. The successful model establishment were confirmed by body weight measurement, behavioral tests, and serum tests. The results showed that the body weight gain of the rats in the stress group was significantly lower than that in the control group; the restraint stress induced an obvious behavioral change and a significantly elevated serum Corticosterone(CORT) and Adrenocorticotropic hormone(ACTH). MK801 administration could alleviate the behavioral changes and serum hormone level dose dependently. The result indicated that chronic restraint stress could result in stressed condition of rats and then ensured the progress of the following experiments.Experiment 2: The effects of psychological stress on the morphology of masseter muscle spindle.In this experiment, we did hematoxylin and eosin staining for the deep masseter muscle tissue. The results showed that psychological stress or MK801 treatment had no significantly effects on the histomorphology of deep masseter. Furthermore, the psychological stress or MK801 treatment also caused no change of muscle spindle number, intrafusal fiber number, intrafusal fiber diameter and capsule wall thickness of deep masseter. The results showed that psychological stress had no obvious effects on the masseter muscle spindle structure in light microscope level.Experiment 3: The effects of psychological stress on the electrophysiological features of masseter muscle spindle afferent.In this experiment, we observed the effects of psychological stress on electrophysiological features of masseter muscle spindle afferent neurons by whole-cell patch clamp technique. The results revealed that psychological stress could significantly decrease the resting membrane potential(RMP), increase the action potential(AP) amplitude, increase the AP decay time, decrease the after hyperpolarization(AHP) amplitude, and increase the firing frequency of muscle spindle afferent neurons. In addition, psychological stress could increase the frequency and amplitude of the spontaneous excitatory postsynaptic current(s EPSC) of the muscle spindle afferent neurons. MK801 treatment could dose-dependently alleviate the changes caused by stress. Our results showed that psychological stress could obviously increase the excitability of muscle spindle afferent neurons whereas MK801 treatment could partly reverse the excitatory effects of stress.Part 2: The mechanisms underlying the effects of psychological stress on masseter muscle afferent in rats.Experiment 1: The effects of psychological stress on the glutamate transporter and its receptors of masseter muscle spindle and masseter muscle spindle afferent neurons.In this part, we observed the effects of psychological stress on the expression of glutamate transporter and glutamate receptors by immunohistofluorescence. The results showed that psychological stress could increase the expression of vesicular glutamate transporter of type 1(VGLUT1) in both peripheral endings(muscle spindle annulospiral endings) and central endings(Vmo); and the Glutaminase 2(GLS2) expression in the cell bodies in Vme was also increased. Moreover, psychological stress obviously increased the expression of VGLUT1-immunoreactive neural endings in Vme as well as NMDAR1, NMDAR2 A, and NMDAR2 B expression in Vme neurons. MK801 had no inhibiting effect on the increased VGLUT1 and GLS2 expression but had potent inhibiting effect on NMDARs expression. The results showed that psychological stress could cause an obvious increase in glutamate synthetize and transportation as well as acceptance of glutamatergic projection in Vme neurons, resulting in enhanced excitability of Vme neurons.Experiment 2: The effects of psychological stress on the synapses structure of masseter muscle spindle afferent neurons.In this part, we observed the effects of psychological stress on the ultrastructure of masseter muscle spindle afferent neurons in Vme by transmission electron microscope examining. Our result showed that psychological stress could increase the number of boutons of excitatory synapses projected to Vme neuron somata, as well as the number of mitochondria in the boutons. The length and thickness of the post synaptic density(PSD) on the post synaptic membrane of excitatory synapses were also increased after stress. In the contrast, the number of boutons, number of mitochondria in the boutons, and the length and thickness of the PSD of inhibitory synapses didn’t changed after stress. MK801 administration could decrease the elevated length and thickness of PSD on the excitatory synapses, but had no effect on the increased number of boutons and mitochondria. The results suggested that psychological stress could enhance the excitability of masseter muscle spindle afferent neurons in Vme via increasing the excitatory synapse projections to the Vme neurons.Conclusion:The psychological stress condition caused by restraint could significantly enhance the excitability of masseter muscle spindle afferent neurons in Vme, resulting a strengthened masseter muscle proprioception transmission into the central neural system. As the main excitatory neurotransmitter of masseter muscle spindle afferent neurons, the synthetize and transportation of glutamate have increased. Meanwhile, psychological stress also increased the glutamatergic projection to Vme neurons somata and the NMDARs expression. In an ultrastructural perspective, the length and thickness of PSD and number of excitatory synapses also increased after psychological stress. On the other hand, NMDARs antagonist MK801 could alleviate the enhanced excitability of masseter muscle spindle afferent neurons in Vme by inhibiting NMDARs expression and reducing the PSD length and thickness. Therefore, the results of the present study could provide a theoretical basis for further in-depth research for the effects of psychological stress on the jaw muscle, the mechanisms underlying it, and the potential therapeutic measures of it.Innovation:1. The present study was the first to explore the effect of experimental induced psychological stress on the morphological and electrophysiological features of masseter muscle spindles.2. The present study was the first to investigate the mechanisms of glutamate and its receptors underlying the increased excitability of masseter muscle spindle afferent neurons caused by experimental induced psychological stress.3. The present study was the first to clarify the involvement of excitatory synapses projecting to Vme neurons in the enhanced excitability of masseter muscle spindle afferent neurons in an ultrastructural perspective.