In recent years gender and sex differences in pain recognition and drug responses have been extensively investigated both in clinical trials and experimental models of pain. Despite a large number of epidemiological studies clearly reveal that women report pain more frequently than men (Unruh, 1996; Berkley, 1997; Riley et al., 1998; Fillingim et al., 2009), results obtained in rodents are not always consistent and often depend on the type of pain and treatment protocol (Craft, 2003; Mogil et al., 2003). More recently, it has been demonstrated that the immune system differently mediates pain hypersensitivity in male and female rodents (Sorge et al., 2015) thus emphasizing the need to elucidate mechanisms underlying sex differences in pain responses with the aim to support the development of gender-specific drug therapies.

Different classes of antidepressant drugs are clinically effective in various forms of chronic pain including arthritis, diabetic neuropathy, post-herpetic neuralgia, migraine, fibromyalgia, low back pain (Gilron et al., 2013). Antidepressant drugs act by blocking the norepinephine and/or serotonin transporter on presynaptic site enhancing the descending inhibitory control on pain (Millan, 2002; Berton and Nestler, 2006; Mika et al., 2013). Nevertheless, many other mechanisms can account for their analgesic activity involving sodium and calcium channels or a number of receptors including GABAB, opioid, and adenosine receptors (Dharmshaktu et al., 2012). In this regard the activity as sodium and calcium blockers as well as the ability to decrease prostaglandin E2 production elicited by several antidepressants including fluoxetine, has been proposed as an important mechanism to explain the efficacy of antidepressants as analgesics (Dharmshaktu et al., 2012).

In clinical practice, it is generally accepted that the tricyclic antidepressant (TCA), amitriptyline, and the serotonin/noreprinephine reuptake inhibitor (SNRI), duloxetine, are effective in persistent pain (Attal et al., 2006; Finnerup et al., 2015). However, there is less agreement about the efficacy of antidepressants belonging to the selective serotonin reuptake inhibitor (SSRI) class (Sindrup et al., 2005; Saarto and Wiffen, 2007; Finnerup et al., 2010). Only few studies have demonstrated some efficacy of SSRIs such as paroxetine and citalopram but not fluoxetine in painful diabetic neuropathy (Sindrup et al., 1990, 1992; Max et al., 1992). On the other hand, in a number of trials that included almost exclusively women, the SSRI fluoxetine, has been shown to be effective in chronic tension-type headache (Walker et al., 1998) and in the treatment of fibromyalgia (Cantini et al., 1994; Wolfe et al., 1994; Goldenberg et al., 1996; Arnold et al., 2002), a musculoskeletal condition characterized by chronic widespread pain and muscle tenderness with a high prevalence on women compared to men (Wolfe et al., 1995).

In rodents the analgesic activity of antidepressant drugs has been evaluated both in models of acute and persistent pain. Most of the studies have been performed with high dose of antidepressants acutely injected (Bomholt et al., 2005; Obata et al., 2005). We have previously shown that fluoxetine is also effective in the formalin test after a single intraperitoneal injection and that the analgesic activity is lost in Lmx1b conditional knock-out mice (Lmx1b^f/f/p), which lack 5-HT neurons in the central nervous system (Zhao et al., 2007). However, protocols of acute treatment do not reproduce the clinical use of these drugs.

Based on these pieces of evidence, we sought to investigate whether a long-term administration with fluoxetine might have sex-specific effects in pain behavior in mice. Moreover, antidepressant treatment may induce direct epigenetic effects and fluoxetine itself is known to modulate the expression of epigenetic modifying enzymes (Perisic et al., 2010; Menke et al., 2012; Zimmermann et al., 2012; Menke and Binder, 2014; Erburu et al., 2015; Vierci et al., 2016).

Epigenetic modifications, including DNA methylation and changes of chromatin structure and function, are the most common mechanisms that control gene expression and, eventually, influence protein expression levels. DNA methylation involves direct chemical modification to the DNA, through the addition of a methyl group at the 5′ position of cytosines in CpG dinucleotides, and usually mediate repression of gene transcription (Newell-Price et al., 2000). On the other hand, a number of post-translational modifications of chromatin structure, including acetylation, methylation, phosphorylation may either increase and repress gene expression (Borrelli et al., 2008). In particular, acetylation is regulated by two classes of enzymes, histone acetyltransferases (HATs) and deacetylases (HDACs) that, respectively, promote acetylation and deacetylation of lysine tails in histone proteins (Mai et al., 2005).

We have first demonstrated that HDAC inhibitors produce analgesic effects via the upregulation of metabotropic glutamate receptor type-2 (mGlu2), an inhibitory receptor whose activation mediates analgesic effects in different pain models (Chiechio et al., 2009). Different HDAC proteins have been identified and subdivided in four classes (I, II, III, and IV) according to their structure, cellular localization and function (Sharma et al., 2013). Class I includes HDAC1, HDAC2, HDAC3, and HDAC8. Class II consists of six HDAC proteins that include HDAC4, HDAC5, HDAC6 HDAC7, HDAC9 and HDAC10, while HDAC11 belongs to class IV. Differently to the other HDAC proteins, class III HDACs operate by a NAD⁺-dependent mechanism and are referred to as sirtuins (SIRT1-7) (Grozinger and Schreiber, 2002). However, among HADACs, the HDAC2 is the only isoform that has been shown to regulate the expression of mGlu2 receptors (Kurita et al., 2012) and fluoxetine itself is known to modulate the expression of HDAC2 (Faillace et al., 2015).

With this in mind, we tested whether a chronic treatment with fluoxetine induces sex specific analgesic effects in the mouse formalin test. We also and evaluated HDAC2 and mGlu2 receptor expression in DRG and dorsal horn by western blot and immunohistochemistry analysis.

Several studies have shown gender differences in antidepressant efficacy (Kokras and Dalla, 2017) and a number of clinical studies have reported that depressed women respond to SSRIs therapy better than males (Haykal and Akiskal, 1999; Kornstein et al., 2000; Quitkin et al., 2002; Berlanga and Flores-Ramos, 2006). Although hormonal and pharmacokintec differences have been postulated as likely candidates to explain different responses to antidepressant pharmacotherapy between sex, the underlying mechanisms are still unknown (Sramek et al., 2016).

Differences in pain sensitivity are also seen in women that usually report greater pain than men (Unruh, 1996; Berkley, 1997; Riley et al., 1998; Fillingim et al., 2009) and antidepressants are effective in relieving chronic pain (Gilron et al., 2013). However, little is known about sex differences in response to chronic antidepressant treatment for the management of chronic pain. Moreover, although it is believed that depression can exacerbate pain, there is evidence that the antidepressant and the analgesic effects occur independently. Antidepressants are analgesic in patients with chronic pain and no concomitant depression (Saarto and Wiffen, 2005, 2007). In addition, the delay of action of antidepressants in chronic pain management in some studies is shorter than in depression (Arnold et al., 2005). This work shows for the first time that chronic administration of fluoxetine induces sex-dependend analgesic effects in mice. We also observed sex-specific changes in mGlu2 expression in the DH and DRGs of female mice together with a decreased expression of HDAC2.

Metabotropic glutamate receptors are known to modulate pain sensitivity (Chiechio and Nicoletti, 2012; Palazzo et al., 2014; Chiechio, 2016). In particular analgesia can be obtained by either blocking group I mGlu receptors, namely mGlu1 and mGlu5 (Walker et al., 2001; Sasikumar et al., 2010; Bennett et al., 2012; Brumfield et al., 2012) or by activating group II and group III mGlu receptors (Chiechio and Nicoletti, 2012; Chiechio, 2016; Johnson et al., 2017). Also, we have demonstrated that epigenetic drugs that increase the expression of mGlu2 receptors are analgesic in models of persistent pain (Chiechio et al., 2002, 2009, 2010; Zammataro et al., 2014).

Antidepressant drugs are known to induce analgesic effects. Different mechanisms have been demonstrated to explain their efficacy in reducing pain. However, long term regulation of gene expression and epigenetic mechanisms are likely to occur after chronic administration. In this regard, it is important to mention that different classes of antidepressant drugs have been demonstrated to have a plethora of effects in CNS disorders and are also known to have a role in neurogenesis (Wang et al., 2011; Masuda et al., 2012; Meneghini et al., 2014; Zhou et al., 2016; King et al., 2017). Many of these effects are sex-dependent (Gray and Hughes, 2015; Kokras and Dalla, 2017) and fluoxetine itself is known to induce sex specific effects on neurogenesis (Hodes et al., 2010). However, so far, no studies have investigated whether long-term treatment with antidepressant drugs affect pain behavior in a sex-dependent manner.

With this study we have shown that chronically administered fluoxetine induces sex-specific analgesic effects in the mouse formalin test and modulates the expression of the epigenetic modifying enzymes, HDAC2, and the expression of mGlu2 receptors in the spinal dorsal horn and DRG of female mice.

A number of HDAC inhibitors have been tested in different pain models and it has been shown that they have analgesic effects through different mechanisms (Chiechio et al., 2009, 2010; Bai et al., 2010; Zhang et al., 2011; Denk et al., 2013; Matsushita et al., 2013; Onofrj et al., 2013; Ximenes et al., 2013; Chen et al., 2014; Kukkar et al., 2014; Zammataro et al., 2014; Capasso et al., 2015; Cao et al., 2016; Tao et al., 2016; Alqinyah et al., 2017; Fork et al., 2017; Lin et al., 2017). We first demonstrated that HDAC inhibitors have analgesic properties via the upregulation of the metabotropic glutamate receptor type-2 (Chiechio et al., 2009, 2010; Onofrj et al., 2013), an inhibitory receptor whose activation or upregulation mediates analgesia (Chiechio et al., 2002, 2009, 2010; Onofrj et al., 2013; Zammataro et al., 2014; Chiechio, 2016). More recently, the isoform HDAC2 has been involved both in pain modulation (Kim et al., 2011; Zhao et al., 2011; Yang et al., 2015; Maiarù et al., 2016) and in the regulation of mGlu2 promoter activity (Kurita et al., 2012) and fluoxetine itself has been reported to regulate the expression of the HDAC2 isoform (Faillace et al., 2015).

In our experimental protocol we did not find sex differences in the formalin test in saline treated mice. Previous studies have reported sex differences in the mouse formalin test only in a late phase, phase 3, developing after 1 h of formalin injection, but not in phases 1 and 2 as observed in the present study (Kim et al., 1999). However, in pain studies phase 3 is rarely measured as it is considered less reproducible and relevant than phases 1 and 2 that reflect acute and tonic phase of pain, respectively (Coderre and Melzack, 1992; Tjølsen et al., 1992). Consistent with our findings, the lack of analgesic effect of chronically injected fluoxetine in male mice has been previously reported in a persistent pain model such as the sciatic nerve cuffing in mice, where, similarly to clinical observations, TCAs such as amitriptyline and nortriptyline were able to elicit analgesic effects while fluoxetine resulted ineffective (Benbouzid et al., 2008). Hormonal and genetic factors might influence pain sensitivity and the response to specific pharmacological treatments. Also, pharmacokinetic differences may account for the analgesic effects of fluoxetine in female mice. Female mice are known to metabolize fluoxetine and to produce more norfluoxetine than males in the brain (Hodes et al., 2010). On the other hand norfluoxetine has been shown to interact with estrogen receptors to regulate ER-mediated gene expression (Lupu et al., 2015). Furthermore, estradiol is able to reduce the expression of HDAC2 in the hippocampus (Zhao et al., 2010).

In summary, this study provides evidence that antidepressant drugs might have sex-specific analgesic effects after chronic administration. The observation that fluoxetine induces sex specific changes in HDAC2 and mGlu2 expression in the dorsal horn of the spinal cord and in DRGs provides a molecular explanation for the analgesic effects in females and suggests that estrogens and/or estrogen receptors might play a role in the observed effects. Studies in ovariectomized female mice and estrogen replacement protocols are in progress to investigate mechanisms underlying the observed differences in male and female mice in response to a chronic treatment with fluoxetine. So far we hypothesize that fluoxetine induces analgesic effects in female mice by reducing the expression of HDAC2 and consequently upregulating the mGlu2 receptor, known to induce analgesia.

All experiments were approved by the local animal ethics committee.

The study was conceived by MZ and SC. Behavioral experiments were designed by MZ, HH, CP, and SC. Immunohistochemistry and western blot experiments were designed by SM, MB, MS, and SC. All authors contributed to writing the manuscript, and all read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.