Estrogen Signaling in Trigeminal Nociception

Abstract

Migraine is much more common in women than in men, and painful episodes are linked to the hormonal fluctuations of the menstrual cycle. The MAP kinase extracellular-signal regulated kinase (ERK) is activated in experimental models of chronic pain, and is also activated by estrogen in sensory neurons. We used an established model of inflammatory trigeminal pain, injection of Complete Freund's adjuvant (CFA) into the masseter muscle, to determine whether ERK activation may play a role in hormone-related trigeminal pain disorders. We measured withdrawal responses to stimulation of the masseter (V3, primary allodynia) and whisker pad (V2, secondary allodynia) using graded monofilaments. Estrogen treatment in the presence of inflammation increased withdrawal response to stimulation of either masseter or whisker pad compared to inflammation alone, indicating an additive effect of inflammation and estrogen on both primary and secondary allodynia. We examined ERK activation in trigeminal ganglia from each treatment group using Western blot and immunohistochemistry. Both masseter inflammation and estrogen treatment increased ERK activation, and combined treatment had an additive effect. Both masseter inflammation and estrogen increased the percentage of pERK immunoreactive neurons in divisions 1 and 2 (V1/2), and combined treatment increased pERK immunoreactivity in V1/2 compared to inflammation alone. We stereotactically administered ERK antagonist U0126, or inactive control U0124, to the trigeminal ganglion of CFA+E2-treated rats. U0126 decreased withdrawal responses to mechanical stimulation of the whisker pad compared to U0124-treated rats. Because the secondary allodynia in V2 after inflammation in V3 was reduced by antagonizing ERK activation in the periphery, these data suggest a peripheral component to secondary trigeminal allodynia mediated through ERK activation. Cellular responses to estrogen can occur through both `genomic' and `nongenomic' pathways. The novel estrogen receptor GPR30 (G-protein coupled receptor 30) activates mediators of signal transduction, including ERK. A goal of this study was to determine which estrogen receptor is required for behavioral sensitization. In order to determine whether GPR30 is present in neurons of the trigeminal ganglion, we performed Western blots for GPR30 on trigeminal ganglion from ovariectomized female Sprague-Dawley rats. Our results show the presence of GPR30 protein in the trigeminal ganglion. Immunohistochemistry for GPR30 in trigeminal ganglion sections showed that GPR30 immunoreactivity was localized to neuronal cell bodies. To determine the subpopulation of neurons that express GPR30, we measured the diameters of GPR30 positive and negative neurons. Neurons expressing GPR30 were significantly smaller in diameter, suggesting that GPR30 is present in nociceptors. In order to investigate GPR30 expression within sub-populations, we co-localized GPR30 with peripherin and NFH using double label immunohistochemistry. GPR30 showed a high degree of overlap with peripherin and partial overlap with NFH, indicating that GPR30 is preferentially expressed in neurons with unmyelinated axons. ERK activation by estrogen in the trigeminal ganglion may occur through either the classical estrogen receptor ERα or through GPR30. In order to determine which estrogen receptor mediates ERK activation in trigeminal ganglion neurons, we examined ERK activation in primary cultures of trigeminal ganglion neurons treated with selective agonists for ERα (PPT), ERβ (DPN), and GPR30 (G-1). ERK was activated by selective agonists for ERα and GPR30, suggesting that activation occurs through either receptor. In order to determine which estrogen receptor mediates increased trigeminal sensitivity, we used a previously employed behavioral model of inflammatory trigeminal allodynia. Ovariectomized female Sprague-Dawley rats were injected in the masseter with CFA and subcutaneously administered G-1,PPT, or vehicle. Withdrawal response to stimulation of the whisker pad (V2) was measured using monofilaments. Either G-1 or PPT treatment increased secondary allodynia, indicating that both receptors function in trigeminal sensitization in vivo. Treatment with estrogen increased expression of ERα but not GPR30, while masseter inflammation increased GPR30 but not ERα. Differential modulation of these ERK-coupled receptors by estrogen and inflammation may play a role in increased trigeminal pain during periods of falling estrogen.