br Experimental procedures br Results

Experimental procedures
Results
Discussion Although previous studies have indicated that astrocyte aromatase plays a role in nociceptive processes (O'Brien et al., 2015, Smeester et al., 2016), the mechanisms responsible for the regulation of aromatase and its involvement in nociception remain unclear. In the present study, we demonstrate three important novel findings. First, sigma-1 receptors and aromatase are colocalized in spinal cord astrocytes. Secondly, the dephosphorylation of aromatase, which is responsible for the rapid activation of aromatase, is associated with formalin-induced acute pain responses. Thirdly, antagonism of sigma-1 receptors blocks the calcineurin-mediated dephosphorylation of aromatase in formalin mice. Collectively these observations suggest that rapidly dephosphorylated astrocyte aromatase plays an important role in the development of formalin-induced acute pain. Furthermore, activated sigma-1 receptors play a critical role in the dephosphorylation of aromatase and this dephosphorylation process is induced through a calcineurin pathway. Aromatase, also called C527 synthase, is the enzyme that converts androgens to estrogens and it is biologically active only at the local tissue level (Simpson and Davis, 2001). Aromatase has previously been shown to be expressed in the spinal cord dorsal horn (Evrard and Balthazart, 2003, O'Brien et al., 2015) in spinal laminae where nociception-sensitive neurons are predominantly located (You et al., 2003). Furthermore, studies using animal pain models have demonstrated that both estrogen receptor-α and β are expressed in high densities in nociceptive neurons (Craft, 2007). These observations are in agreement with the studies showing that estrogen can alter the intrinsic and synaptic physiology of neurons via a PKCε-dependent pathway, producing acute pain sensation (Hucho et al., 2006, Woolley, 2007, Ji et al., 2011). LET is a non-steroidal aromatase inhibitor that effectively blocks aromatase activity without interfering with other steroid biosyntheses (Elisaf et al., 2001). In the current study, we examined whether the inhibition of aromatase with LET significantly reduced formalin-induced nociceptive behavior and associated Fos expression in the spinal cord dorsal horn. In this regard it is important to note that the hindpaw injection of formalin produces a biphasic nociceptive response; the first phase is thought to result from direct activation of c-fiber primary afferents, while the second phase is thought to signify both the effects of afferent input and spinal cord central sensitization. Thus, our results showing that LET reduces paw licking during the second phase of the formalin test suggests that activated aromatase plays a role in the development of more tonic pain processes in formalin mice. Several studies have reported that brain or spinal cord aromatase is expressed in neurons, suggesting that locally synthesized estrogen may affect a variety of extrinsic and intrinsic neuronal circuits (Yague et al., 2006). However, several recent papers, as well as, the present results have demonstrated that aromatase is expressed in astrocytes, but not in neurons, in the spinal cord (O'Brien et al., 2015). There are several possible reasons for this discrepancy. It has been reported that the cellular location of aromatase in the CNS can be diverse depending on the animal species, age and sex, as well as the region of the brain under study (Yague et al., 2006, Stanic et al., 2014, Prange-Kiel et al., 2016). Furthermore, under various pathological states, such as spinal cord injury or amyotrophic lateral sclerosis, astrocytes also express aromatase, suggesting that certain pathologies may induce a glial enzyme expression pattern (Garcia-Segura et al., 1999, Sun et al., 2017). The antibody used in the present study is specific for aromatase and under the conditions used in our immunohistochemical protocol specifically stains spinal astrocytes in male ICR mice. As suggested above, variations in reports of aromatase localization to astrocytes versus neurons in the literature may be due to species and/or strain differences, the age and sex of the animals, antibody specificity or to the unique pathological state of the animal.