Relationship of Spinal Dynorphin Neurons to -Opioid Receptors and Estrogen Receptor : Anatomical Basis for Ovarian Sex Steroid Opioid Antinociception

Pharmacological and behavioral studies suggest that spinal and -opioid antinociceptive systems are functionally associated with ovarian sex steroids. These interactions can be demonstrated specifically during pregnancy or hormone-simulated pregnancy (HSP). The analgesia associated with both conditions can be abolished by blockade of either spinal -opioid receptors or -opioid receptors (DOR). Furthermore, both dynorphin (DYN) release (J Pharmacol Exp Ther 298:1213– 1220, 2001) and the processing of the DYN precursor (J Neurochem 65:1374–1380, 1995) are significantly increased in the spinal cord during HSP. We undertook the current study to determine whether DYN, DOR, and estrogen receptor (ER ) share anatomical relationships that permit their direct interaction. Coexpression of DOR or ER by DYN neurons was assessed using fluorescence immunohistochemistry and a synaptosomal release assay. Findings show that ER and DYN are coexpressed. Moreover, in the spinal cord of HSP animals, there were significant increases in the number of DYN-immunoreactive (DYN-ir) cells, ER -ir cells, cells double-labeled for DYN-ir and ER -ir and the proportion of DYN-ir cells coexpressing ER . Some varicose fibers in the spinal cord dorsal horn and intermediate gray matter that expressed DYN-ir also expressed DOR-ir. Activation of DORs located on DYN terminals was sufficient to inhibit K -evoked DYN release. These data define, at least in part, the anatomical substrates that may be relevant to the antinociception of gestation and its hormonal simulation. Furthermore, they provide a framework for understanding sex-based nociception and antinociception and suggest novel strategies for treating pain. Pharmacological and behavioral studies have suggested a functional association between spinal and -opioid antinociceptive systems and their modulation by ovarian sex steroids (Dawson-Basoa and Gintzler, 1998). Response thresholds to somatic and visceral noxious stimuli are elevated during gestation (gestational antinociception, GSA) in rats, mice, sows (Toniolo et al., 1987; Gintzler and Bohan, 1990; Iwasaki et al., 1991), and humans (Cogan and Spinnato, 1986). Simulation of the concentration profiles of 17 -estradiol (estrogen, E2) and progesterone (P) found in the blood during pregnancy (i.e., hormone-simulated pregnancy, HSP) also increases thresholds for responsiveness to aversive stimuli (HSP analgesia, HSPA). Moreover, the magnitude, temporal pattern of onset, and duration, and spinal opioid receptor profile of HSPA are essentially identical to those of physiological pregnancy (Dawson-Basoa and Gintzler, 1993, 1998; Liu and Gintzler, 1999). In rats, pharmacological blockade of either spinal -opioid receptors (KOR) or -opioid receptors (DOR) abolishes GSA and HSPA (Dawson-Basoa and Gintzler, 1998; Liu and Gintzler, 2000). This indicates that ovarian sex steroids can induce activation of spinal KOR and DOR analgesic systems and that both of these effects are essential for the manifestation of GSA and HSPA. Functional interactions between spinal DOR and KOR pathways are also indicated by neurochemical analyses. Exposure of nonpregnant ovariectomized animals to pregnancy levels of E2 and P (E2/P) results in a 1 Current affiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.139816. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: GSA, gestational antinociception; DYN, dynorphin; DYN-ir, dynorphin immunoreactivity; ER , estrogen receptor ; ER -ir, estrogen receptor immunoreactivity; HSP, hormone-simulated pregnancy; HSPA, HSP analgesia; DOR, -opioid receptor; DOR-ir, -opioid receptor immunoreactivity; E2, 17 -estradiol, estrogen; P, progesterone; KOR, -opioid receptor; NA, numerical aperture; DPDPE, D-Pen enkephalin; PBS, phosphate-buffered saline. 0022-3565/08/3263-725–731$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 326, No. 3 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 139816/3373370 JPET 326:725–731, 2008 Printed in U.S.A. 725 http://jpet.aspetjournals.org/content/suppl/2008/06/09/jpet.108.139816.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Sptem er 2, 2017 jpet.asjournals.org D ow nladed from significant increase in spinal cord processing of DYN precursor intermediates (Medina et al., 1995) and in both basal and stimulated rates of lumbar dynorphin release (Gupta et al., 2001). These findings suggest the presence of sex steroid receptors on dynorphin somata and/or terminals. Strikingly, whereas activation of spinal DOR inhibits the stimulated dynorphin release from spinal tissue of control females, DOR activation facilitates evoked DYN release from spinal tissue of HSP animals, suggesting that sex steroid-initiated signaling events can change the responses of spinal DYN neurons to DOR activation. Although it has been well established that both GSA and HSPA require the participation of DOR, DYN, and ovarian sex steroids, the spinal organization supporting interactions among them has not been elucidated. The most parsimonious anatomical relationship among DOR, DYN, and ER consistent with their role in GSA and HSPA would be one that allows their direct interaction. Accordingly, we hypothesized that 1) spinal DOR and/or ER are coexpressed by DYN neurons and that 2) their expression is affected by ovarian sex steroids. These hypotheses were tested in HSP, a condition that closely mimics the blood concentration profile of E2/P that occurs during gestation (Bridges, 1984) but avoids the potential confounds of the other aspects of pregnancy, e.g., changes in sensory input due to uterine distension. Materials and Methods Experimental Animals. Experiments with HSP or control rats employed female Sprague-Dawley rats (Charles River, Kingston, NY; 250–300 g) that were maintained in an approved controlled environment on a 12-h light/dark cycle. Food and water were available ad libitum. All experimental procedures were reviewed and approved by the Animal Care and Use Committees of State University of New York Downstate Medical Center. Induction of HSP. E2/P was administered via the subcutaneous implantation of silicone elastomer (Silastic; Dow Corning, Midland, MI) tubing filled with either a solution of E2 (in sesame oil) or crystalline P (Bridges, 1984) as routinely employed by this laboratory (Dawson-Basoa and Gintzler, 1993). Controls consisted of implants containing sesame oil (vehicle for E2) and empty silicone elastomer tubing (as a vehicle control for P). Day 1 of steroid hormone administration or its vehicle control was initiated at the time