**1.3 Estrogen receptors and nociceptive signaling in primary afferent neurons**

Defining the site(s) and mechanisms through which sex estrogen modulates visceral nociception is an important step in understanding the mechanisms in pain perception associated with endometriosis and in designing appropriate therapies. One such mechanism may be the convergence of nociceptive stimuli and estrogen input on the primary afferent neurons which innervate viscera (i.e. uterus). Estrogen may modulate female sensitivity to clinical and experimentally induced pain. Based on our preliminary results, it is likely that estrogen receptors (ERs) expressed in primary afferent neurons modulate chemical signaling associated with nociception. Nociception is a balance of pro- and anti-nociceptive inputs that is subject to regulation depending on the normal state of the organism. Sensitization of primary afferent neurons to stimulation may play a role in the enhanced perception of visceral sensation and pain. Chest pain from coronary heart disease, endometriosis, acute and recurrent/chronic pelvic pain in women or abdominal are all visceral pain sensations that may result in part from sensitization (Berkley *et al.* 2001; Mayer *et al.* 2001). Mechanisms of peripheral sensitization may involve increased transduction that is secondary to repeated stimulation or an increase in the excitability of the afferent nerves by molecules that decrease the excitation threshold (Zimmermann 2001)

The cell bodies of primary visceral spinal afferent neurons are located in DRG. Direct activation of chemosensitive receptors and ion channels on their peripheral terminals and modulation of neuronal excitability activates extrinsic primary afferent nerves. Nociceptors belong predominantly to small and medium size DRG neurons whose peripheral processes detect potentially damaging physical and chemical stimuli. The terminals of primary visceral afferent neurons are described as having no organs end or morphological specialization, but respond to different chemical stimuli. Visceral nociceptive C-fibers activated by ATP released by noxious stimuli from cells in target organs, have been implicated as mediators of noxious stimulus intensities (Burnstock 2000). Alteration in signal transduction of primary afferent neurons can result in enhanced perception of the visceral sensation that is common in patients with different disorders resulting in elevated pain perception. Acute and recurrent/chronic pelvic pain in women and abdominal pain from IBS are illustrative examples of visceral pain that undergo sensitization (Giamberardino *et al.* 2010).

Peripheral sensitization can develop in response to sustain stimulation, inflammation, and nerve injury. Visceral pain is different from cutaneous pain based on clinical,

Dorsal root ganglion (DRG) neurons can be activated or modulated by the activation of chemosensitive receptors on peripheral terminals and ATP has been implicated in sensory transduction of noxious stimuli by activating purinergic P2X receptors (Dunn *et al.* 2001). Once released into the intercellular areas, the action of ATP is mediated by primarily P2X3 receptors which are expressed on primary afferent fibers and cell bodies within DRG (Burnstock 2001). The capsaicin-sensitive primary afferent neurons of small- and mediumdiameter neurons mediate nociceptive-like behaviors suggesting that TRPV1 expressing neurons are nociceptors. Activation of purinergic (P2X3) and transient potential receptors family vcanilloid-1 (TRPV1) receptors results in the depolarization and opening of voltagegated Ca2+channels (VGCC) (Koshimizu *et al.* 2000). A sensation of pain is produced by

**1.3 Estrogen receptors and nociceptive signaling in primary afferent neurons** 

molecules that decrease the excitation threshold (Zimmermann 2001)

(Giamberardino *et al.* 2010).

Defining the site(s) and mechanisms through which sex estrogen modulates visceral nociception is an important step in understanding the mechanisms in pain perception associated with endometriosis and in designing appropriate therapies. One such mechanism may be the convergence of nociceptive stimuli and estrogen input on the primary afferent neurons which innervate viscera (i.e. uterus). Estrogen may modulate female sensitivity to clinical and experimentally induced pain. Based on our preliminary results, it is likely that estrogen receptors (ERs) expressed in primary afferent neurons modulate chemical signaling associated with nociception. Nociception is a balance of pro- and anti-nociceptive inputs that is subject to regulation depending on the normal state of the organism. Sensitization of primary afferent neurons to stimulation may play a role in the enhanced perception of visceral sensation and pain. Chest pain from coronary heart disease, endometriosis, acute and recurrent/chronic pelvic pain in women or abdominal are all visceral pain sensations that may result in part from sensitization (Berkley *et al.* 2001; Mayer *et al.* 2001). Mechanisms of peripheral sensitization may involve increased transduction that is secondary to repeated stimulation or an increase in the excitability of the afferent nerves by

The cell bodies of primary visceral spinal afferent neurons are located in DRG. Direct activation of chemosensitive receptors and ion channels on their peripheral terminals and modulation of neuronal excitability activates extrinsic primary afferent nerves. Nociceptors belong predominantly to small and medium size DRG neurons whose peripheral processes detect potentially damaging physical and chemical stimuli. The terminals of primary visceral afferent neurons are described as having no organs end or morphological specialization, but respond to different chemical stimuli. Visceral nociceptive C-fibers activated by ATP released by noxious stimuli from cells in target organs, have been implicated as mediators of noxious stimulus intensities (Burnstock 2000). Alteration in signal transduction of primary afferent neurons can result in enhanced perception of the visceral sensation that is common in patients with different disorders resulting in elevated pain perception. Acute and recurrent/chronic pelvic pain in women and abdominal pain from IBS are illustrative examples of visceral pain that undergo sensitization

Peripheral sensitization can develop in response to sustain stimulation, inflammation, and nerve injury. Visceral pain is different from cutaneous pain based on clinical,

depolarization of the peripheral nerve terminals.

neurophysiological and pharmacological characteristics (Chang and Heitkemper 2002). The pathophysiology of visceral hyperalgesia is less well-known than its cutaneous counterpart, and our understanding of visceral hyperalgesia is colored by comparison to cutaneous hyperalgesia, which is believed to arise as a consequence of the sensitization of peripheral nociceptors due to long-lasting changes in the excitability of spinal neurons. Endometriosis is currently defined as a chronic functional syndrome characterized by recurring symptoms of abdominal discomfort or pain. In the context of visceral pain, the TRPV1 receptor is a sensory neuron-specific cation channel which plays an important role in transporting thermal and inflammatory pain signals. Evidence for TRPV1's role is that mice lacking TRP1 receptor gene have deficits in thermal- or inflammatory-induced hyperalgesia (Davis *et al*. 2000). Activation of both TRPV1 and P2X receptors induce mobilization of [Ca2+]i in cultured DRG neurons (Gschossmann *et al.* 2000).

Sex hormones and 17β-estradiol (E2) in particular may directly influence the functions of primary afferent neurons since both ERs are present on small-diameter DRG neurons (Papka and Storey-Workley 2002). Despite the broad spectrum of E2 effects in the nervous system, the mechanisms of hormonal pain modulation remain unclear. There are two subforms: estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) which were traditionally thought of as ligandactivated transcription factors. However, recent work has demonstrated multiplicity of E2 actions (membrane, cytoplasmic and nuclear) (Nadal *et al*. 2001). ER distributed through CNS and PNS including regions that mediate nociception. For example, ERs are expressed in dorsal horn neurons of the spinal cord and DRG neurons. DRG neurons express both ERα and ERβ *in vivo* (Papka and Storey-Workley 2002) and *in vitro* (Chaban 2010). These findings suggest that E2 may modulate sensory input at the primary afferent level. E2 can alter gene transcription, resulting in pro-nociceptive (reducing β-endorphin expression) or anti-nociceptive (increasing enkephalin expression) changes of endogenous opioid peptides , opioid receptors (Micevych and Sinchak 2001) and, by increasing levels of CCK, an anti-nociceptive and anti-opioid molecule (Micevych *et al.* 2002).

E2 can modulate cellular activity by altering ion channel opening and second messenger signaling by stimulating G-proteins (Chaban *et al*. 2003) , the signal transduction pathways traditionally associated with membrane receptor activation. Many of these effects have been ascribed to membrane-associated receptors. The results from other laboratories (Lee *et al.* 2002) and our data (Chaban *et al*. 2003) indicate that E2 is acting to modulate L-type VGCC. The cloned TRPV1 receptor is a nonselective cation channel with a high permeability for Ca2+. TRPV1's are distributed in peripheral sensory nerve endings and are involved in the transduction of different stimuli in sensory neurons. TRPV1 functions as molecular integrator of painful chemical and physical stimuli (noxious heat (>43º C) and low pH). Various inflammatory mediators such as prostaglandin E2 (PGE2) and bradykinin potentiate TRPV1. The potentiation of TRPV1 activity can be quantified by measuring the differences of capsaicin-induced Ca2+ concentration changes before and after receptor activation (Petruska *et al*. 2000). Significantly, a subset of DRG neurons respond to both capsaicin and ATP indicating that there may be cross-activation of these receptors that may underlie the sensitization of visceral nociceptors. Capsaicin-induced TPRV1 receptor-mediated changes in [Ca2+]i may represent a level of DRG activation to noxious cutaneous stimulation while ATP-induced changes in [Ca2+]i may reflect the level of DRG neuron sensitization to noxious visceral stimuli since ATP is released by noxious stimuli and tissue damage near the primary afferent nerve terminals (Burnstock 2001).

Primary Afferent Nociceptors and Visceral Pain 373

Care and Use committee at the University of California and the NIH Guide for the Care and

Experiments were performed on age-matched (8–10 wk old) heterozygous mutant mice lacking the gene male (ERα−*/*−) and female (ERα−*/*−) for ERα (ERα−*/*−), and the deficiency ERβ (ERβ−*/*−) mice were bred into heterozygous mutant female mice (ERβ−*/*−) and homozygous male mutant mice (ERβ−*/*−) (Jackson Laboratory, Bar Harbor, ME, USA). Mice were housed in climate-controlled rooms, and standard rodent chow and water were available *ad libitum* and were housed in accordance with the NIH Guide for the Care and

The isolation procedure and primary culture of mouse lumbosacral DRG has been published in detail (Chaban, Mayer et al. 2003). DRG tissues were obtained from c57/black 6J (The Jackson Laboratory; 30 g), ERαKO and ERβKO (Taconic; 20 g) transgenic types. Briefly, lumbosacral adult DRGs (level L1-S1) from Wt, ERαKO and ERβKO mice will be collected under sterile technique and placed in ice-cold medium Dulbecco's Modified Eagle's Medium (DMEM; Sigma Chemical Co., St. Louis, MO). Adhering fat and connective tissue will be removed and each DRG will be minced with scissors and place immediately in a medium consisting of 5 ml of DMEM containing 0.5 mg/ml of trypsin (Sigma, type III), 1 mg/ml of collagenase (Sigma, type IA) and 0.1 mg/ml of DNAase (Sigma, type III) and kept at 37°C for 30 minutes with agitation. After dissociation of the cell ganglia, soybean trypsin inhibitor (Sigma, type III) will be used to terminate cell dissociation. Cell suspension will be centrifuged for one minute at 1000 rpm and the cell pellet will be resuspended in DMEM supplemented with 5% fetal bovine serum, 2 mM glutamine-penicillin-streptomycin mixture, 1 μg/ml DNAase and 5 ng/ml NGF (Sigma). Cells will be plated on Matrigel® (Invitrogen)-coated 15-mm coverslips (Collaborative Research Co., Bedford, PA) and kept at 37° C in 5% CO2 incubator for 24 hrs, given fresh media and maintained in primary culture

The expressions of TRPV1 and of P2X3 receptors in L1~S1 DRGs were studied by using Western blot analyses. Tissues from wild type (C57BL/6J), ERαKO, and ERβKO mice were quick frozen in tubes on dry ice during collection. L1~S1 DRG were combined, homogenized by mechanical disruption in ice-cold RIPA buffer plus protease inhibitors and incubated on ice for 30 minutes. Homogenates were then spun at 5000 g for 15 minutes and supernatants collected. Total protein was determined on the supernatants using the BCA microtiter method (Pierce, Rockford, Ill., USA). Samples containing equal amounts of protein (40µg) were electrophoresed under denaturing conditions using Novex Mini-cell system (San Diego, Calif., USA) and reagents (NuPage 4–12% Bis-Tris gel and MOPS running buffer). After electrophoretic transfer onto nitrocellulose membrane using the same system, the membrane was blocked with 5% non-fat dry milk (NFDM) in 25 mM TRIS buffered saline, pH 7.2, plus Tween 20 (TBST) for 1 hour at room temperature, followed by incubation with polyclonal rabbit antibody against TRPV1-N terminus (1:1000, Neuromics) and P2X3 receptor (1:1000, Neuromics) for overnight at 4oC. The membrane was then

Use of Laboratory Animals.

Use of Laboratory Animals

**2.3 Primary culture of DRG neurons** 

until used for experimental procedures.

**2.4 Western blot analysis** 

**2.2 Animal breeding** 

Most of the published reports about sex and hormone-related differences in pain have addressed the modulatory effect of E2 on central nervous system mechanisms of nociception (Aloisi *et al.* 2000). Recent studies demonstrate that E2 has a significant role in modulating viscerosensitivity, indicating that E2-induced alterations in sensory processing may underlie sex-based differences in functional pain syndromes (Al-Chaer and Traub 2002). However, reports of E2 modulation of visceral and somatic nociceptive sensitivity are inconsistent. For example, elevated E2 levels have been reported to increase the threshold to cutaneous stimuli but decrease the percentage of escape responses to ureteral calculosis (Bradshaw and Berkley 2002). Additionally, nociceptive sensitivity increases when E2 levels are elevated (Holdcroft 2000; Bereiter 2001). Indeed in most clinical studies, women report more severe pain levels, more frequent pain and longer duration of pain than men. To help resolve these inconsistencies we propose to study E2 actions on the primary afferents.

Primary DRG neurons culture has been a useful model system for investigating sensory physiology and putative nociceptive signaling (Chaban *et al*. 2003). ATP-induced intracellular calcium concentration ([Ca2+]*i*) transients in cultured DRG neurons have been used to model the response of nociceptors to painful stimuli. In our laboratory we showed that E2, acting at the level of the plasma membrane, attenuates both ATP -induced [Ca2+]<sup>і</sup> and capsaicin- induced [Ca2+]i influx and that the expression of both P2X3 and TRPV1 depend on the expression of both ERs. Within the context of our hypothesis visceral nociception and nociceptor sensitization appear to be regulated by P2X3 and TRPV1. Estrogen attenuates DRG neurons response to ATP and capsaicin suggesting that visceral afferent nociceptors can be modulated by sex steroids at a new site at the level of primary afferent neurons. Our data suggest that E2 by itself appears to be anti-nociceptive but interferes with anti-nociceptive actions of other pain-modulating drugs (such as opioids). Thus, E2 acting on primary afferent nociceptors modulates the response to pro- and antinociceptive signals**.** Within the context of our cross-sensitization hypothesis, inflammation sensitizes non-inflamed viscera that are innervated by the same DRG and/or crosssensitization occurs as a result of intra-DRG release of sensitizing mediators such as ATP or substance P in the DRG (Matsuka *et al.* 2001; Chaban 2008; Chaban 2010).

Lumbosacral DRG neurons (levels L6-S1) from wild type mice (WT) express estrogen receptors (ERα and ERβ), purinergic P2X3, vanilloid TRPV1, SP and methabotropic glutamate (mGluR2/3) receptors. In our recent studies we also tested the difference in how somatic and visceral afferents are modulated by E2. Both short-term and long-term exposure to E2 significantly decreased the ATP and capsaicin-induced increase in [Ca2+]i.
