Psychophysical stress increases the expression of phospho-CREB, Fos protein and neurokinin-1 receptors in superficial laminae of trigeminal subnucleus caudalis in female rats

Abstract

Psychological stress and estrogen status are risk factors to develop painful temporomandibular joint disorders (TMJD); however, the neural basis for this relationship is not known. This study tested the hypothesis that repeated forced swim stress and estradiol treatment alter the phosphorylation of cAMP responsive element-binding protein (pCREB) in trigeminal subnucleus caudalis (Vc), the initial site of sensory input from the TMJ. Ovariectomized female rats were given low or high dose estradiol and subjected to repeated forced swim stress for 3 days and on day 4 an intra-TMJ injection of mustard oil or vehicle was given. Forced swim alone increased the number of pCREB-positive neurons, independent of estradiol treatment or TMJ stimulation, in superficial and deep laminae of Vc. Forced swim also increased the number of Fos-positive neurons in superficial laminae and neurokinin-1 receptor mRNA in whole dorsal Vc, independent of estradiol treatment. These results indicated that persistent psychophysical stress alone was sufficient to increase the expression of pCREB and downstream regulated genes associated with enhanced excitability in the caudal medullary dorsal horn, a brainstem region thought to be critical for TMJD pain.

Figure 1

Repeated FS conditioning reduces maximum forelimb grip force. A. Grip force was reduced similarly in LE- and HE-treated rats. Sample sizes: LE = 12; HE = 14. *p < 0.05, **p < 0.01 versus day 1 value. B. Reduction in grip force after FS was inversely correlated with the number of pCREB-positive neurons in superficial laminae. n = 26.

Figure 2

Examples of pCREB staining at the Vc/C_1-2 junction in LE-treated rats. A. Control. B. Repeated FS condition. Boxed areas represent the superficial and deep laminar regions for cell counting (640 μm²). Calibration = 150 μm.

Figure 3

Summary of pCREB cell counts at the Vc/C_1-2 junction. A. superficial laminae. B. deeper laminae. Abbreviations: E2, estradiol benzoate; FS, forced swim; HE, high E2; LE, low E2; MO, mustard oil. *p < 0.05, **p < 0.01 ipsilateral versus contralateral; a = p < 0.05, b = p < 0.01 0% versus 20% MO for the equivalent E2-treated non-FS rats; ## = p < 0.01 versus corresponding sham swim groups. n = 4-6 rats per group.

Figure 4

Repeated FS increases the number of Fos-positive neurons in superficial laminae at the Vc/C_1-2 junction (A) and NK-1 mRNA in whole Vc/C_1-2 tissue samples (B). a = p < 0.05, b = p < 0.01 versus sham swim groups. n = 4-6 rats per group.