This study aimed to determine whether psychophysical stress conditionings had facilitatory effects on masseter muscle nociception in the central nervous system via serotonergic mechanisms in rats. Two experiments were conducted to assess: (1) whether repeated forced swim stress for 3 days increased the number of Fos-positive neurons evoked by masseter muscle injury due to formalin injection; and (2) whether serotonin-reuptake inhibitor, fluoxetine, administered daily after each stress conditioning, had modulatory roles on Fos expression. The number of Fos-positive cells was quantified in several areas within the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord regions (Vc areas), including the ventrolateral area of the trigeminal subnucleus interpolaris/Vc transition, and the middle or caudal portion of the Vc regions, since nociceptive neural activity in the Vc region could play critical roles in deep craniofacial nociception. We found that forced swim stress conditionings increased depression-like behaviors, which was prevented by fluoxetine. Repeated forced swim stress significantly increased Fos expression in all Vc areas compared with those of non-stressed rats, while systemic administration of fluoxetine significantly decreased Fos expression in all areas, but mainly in the caudal Vc region, in stressed rats. Fluoxetine had no effect on Fos expression in non-stressed rats. These results indicate that repeated forced swim stress conditionings increase Fos expression in the Vc areas, and the contribution of serotonergic mechanisms to masseter muscle nociception could be greater in stressed rats than in sham rats. These results support the hypothesis that changes in brain function, including serotonergic mechanisms, in the Vc areas play critical roles in enhanced masseter muscle nociceptive responses under psychophysical stress conditions.

Psychophysical stress can cause neural changes that increase nociception in the orofacial region, particularly the masseter muscle (MM). The nucleus raphe magnus (NRM), which is located in the brain stem, serves the crucial role of regulating nociception through descending modulatory pain control. However, it remains unclear if neural activities in the NRM are affected under psychophysical stress conditions. This study conducted experiments to assess (1) whether neural activity, indicated by Fos expression in an NRM that has experienced MM injury, is affected by the stress of repeated forced swim tests (FST); and (2) whether the selective serotonin reuptake inhibitor fluoxetine administered daily after an FST could affect the number of Fos-positive neurons in the NRM. Results revealed that the stress from repeated FSTs significantly increased the number of Fos-positive neurons in an NRM that had been affected by MM injury. Fluoxetine inhibited increases in the number of Fos-positive neurons in the NRM that occurred as a result of FSTs, but this was not observed in sham rats. These findings indicate that the stress from FSTs could increase nociceptive neural activity in an NRM that has experienced MM injury. This could be due, in part, to changes in serotonergic mechanisms.

We tested whether Sake Lees (SL) had inhibitory effects on hyperalgesia in the hindpaw under psychophysical stress conditions. Male rats were subjected to repeated forced swim stress treatments (FST) from Day −3 to Day −1. Intraperiotoneal administration of SL which contained low concentration of ethanol (SLX) was conducted after each FST. On Day 0, formalin-evoked licking behaviors and Fos responses in the lumbar spinal cord (DH) and several areas within the rostral ventromedial medulla (RVM) were quantified as nociceptive responses. FST-induced hyperalgesia in the hindpaw was prevented by repeated SL and SLX treatments. Fos expression was significantly increased in DH and some areas within the RVM under FST, which was prevented by repeated SL or SLX. These findings indicated that daily administration of SL had the potential to alleviate stress-induced hyperalgesia.

We determined if Japanese Rice Wine (Sake) had inhibitory effects on stress-induced enhancement of masseter muscle (MM) nociception in the rats. Male rats were subjected to the repeated forced swim stress (FS) or sham conditionings from Day −3 to −1. Daily administration of Sake or saline was conducted after each stress conditioning. At Day 0 the number of Fos positive cells, a marker for neural activity, was quantified at the trigeminal subnucleus caudalis (Vc) region by MM injury with formalin. FS increased MM-evoked Fos expression in the Vc region, which was inhibited by Sake compared to saline administration. Sake did not alter the number of Fos positive cells under sham conditions, indicating that inhibitory roles of Sake on neural activity in the Vc region were seen under FS conditions. These findings indicated that Sake had inhibitory roles on stress-induced MM nociception at the Vc region in our experimental conditions.

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