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NEUROPHARMACOLOGY

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

Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, New York (A.R.G., D.S.G., N.-J.L.) and Department of Neuroscience (S.A.S., M.W.W.), University of Minnesota, Minneapolis, Minnesota

Received for publication April 9, 2008
Accepted June 4, 2008.

	Abstract

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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.

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 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

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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 of ovariectomy.

Tissue Preparation for Immunohistochemistry. Rats were deeply anesthetized with a mixture of ketamine (68 mg/kg), xylazine (4.6 mg/kg), and acepromazine (0.9 mg/kg). Rats were perfused through the ascending aorta with 100 ml of ice-cold oxygenated calcium-free Tyrode's buffer (115 mM NaCl, 5 mM KCl, 2 mM MgCl₂·6H₂O, 400 µM MgSO₄·H₂O, 3 mM glucose, and 25 mM NaHCO₃, pH 7.2) followed by 500 ml of freshly prepared buffered formaldehyde (4% w/v formaldehyde, 14% v/v saturated aqueous picric acid, 75 mM KH₂PO₄, and 85 mM Na₂HPO₄·7H₂O, pH 6.9). After fixation, the entire vertebral column, including the pelvis, was harvested, placed in cryoprotectant solution (15 mM sucrose, 30 mM K₂HPO₄, and 70 mM Na₂HPO₄·H₂O, pH 7.2), and stored at 4°C until it was shipped from New York to Minnesota via overnight air courier service.

Preparation and Superfusion of Spinal Cord Synaptosomes. The spinal vertebral column was sectioned at the intervertebral spaces above vertebrae T₁₂ and L₁. The lumbar spinal cord contained within this segment (L₁–L₅; 200–250 mg) was quickly expelled by injecting ice-cold saline into the caudal end. Lumbar spinal cord was homogenized in 10 ml of ice-cold 0.32 M sucrose and 5 mM Tris, pH 7.4, using a polytetrafluoroethylene (Teflon; DuPont, Wilmington, DE) glass homogenizer (15 strokes). Homogenate was centrifuged at 1000g for 5 min. The supernatant thus obtained was centrifuged at 15,000g for an additional 20 min. The resulting pellet (P2) was resuspended in 300 µl of pregassed (95% O₂ and 5% CO₂) Krebs buffer [containing the protease inhibitors captopril (10 µM), thiorphan (0.3 µM), bestatin (10 µM), and L-leucyl-L-leucine (2 mM)], 200 µl of which was layered over 100 µl of Sephadex P-10 slurry (60%; Pharmacia Fine Chemicals, Uppsala, Sweden) that had been added to a superfusion chamber containing a Whatman GF/B filter (Whatman, Ampark, NJ) at its outlet. The chamber was superfused with Krebs buffer using a SF-06 Suprafusion apparatus (Brandel, Gaithersburg, MD). After a 40-min preincubation, basal release was assessed over two 6-min collections, after which 50 mM K⁺-evoked release was evaluated over 3 min. Subsequently, two additional cycles of release were collected, each separated by a 15-min incubation. The second release cycle was collected in the presence of the -opioid agonist D-Pen², D-Pen⁵-enkephalin (DPDPE) (1 µM), after which the conditions for the first cycle were repeated. For all of the reported data, basal and evoked release in cycles 1 and 3 were indistinguishable.

Dynorphin Radioimmunoassay. Superfusates containing basal release and evoked release were desalted and concentrated using reverse-phase C18 Maxi-Clean cartridges (Alltech, Deerfield, IL). DYN peptide, eluted with 70% acetonitrile/0.1% trifluoroacetic acid, was lyophilized to dryness and stored at 4°C. Recovery of DYN A-(1–17) was quantitative (95%). DYN content was assessed using a scintillation proximity radioimmunoassay. The standard DYN peptide or the DYN extracted from suprafusate was resuspended in 110 µl of 0.1 M sodium phosphate (0.1% BSA) and incubated with 20 µl (3 µg/ml) of purified rabbit anti-DYN antibody (Peninsula Laboratories, San Carlos, CA) for 2 h at room temperature, after which ¹²⁵I-labeled DYN A (Bachem Biosciences, King of Prussia, PA; 10,000 cpm) was added and allowed to incubate for an additional 2 h at room temperature. Fifty microliters of anti-rabbit scintillation proximity polyvinyl toluene beads (Amersham Biosciences, Buckinghamshire, UK; 1 mg/50 µl in 0.1 M phosphate buffer) was added and incubated overnight at room temperature on a circular shaker. The next day, the assay mixture was transferred to a 96-well polystyrene clear plate for counting using a MicroBeta JET Counter (PerkinElmer Life and Analytical Sciences, Waltham, MA). The minimum detectable concentration of DYN is 1.9 pg, with an ED₅₀ of 22 pg.

Immunohistochemistry. The L₅, L₆, S₁, and S₂ spinal segments were identified and mounted for sectioning. Tissue was quickly frozen, and sections were cut at a nominal thickness of 10 µm on a cryostat (Bright Instruments, Huntington, UK), thawed onto Fisher-finest Capillary gap slides (Thermo Fisher Scientific, Waltham, MA), and stored at -20°C until used. To minimize the effects of variations in staining conditions and thus maximize the reliability of our comparisons, spinal cord from two control and two experimental animals were embedded in the same block. This allowed sections from each of the four tissue samples, which could be distinguished by their orientation, to be cut, mounted, and processed concomitantly. Sections were rinsed in PBS (140 mM NaCl, 10 mM Na₂HPO₄, 3 mM KCl, and 2 mM KH₂PO₄, pH 7.4) and then coincubated overnight at room temperature with a 1:300 dilution in PBS containing 0.3% Triton X-100 of guinea pig anti-preprodynorphin-(235–248). Preprodynorphin-(235–248) is a cryptic portion of the DYN precursor that serves as a marker peptide for DYNergic neurons (Arvidsson et al., 1995b). One of two other antibodies was added to the anti-preprodynorphin solution, either a 1:1000 dilution of rabbit anti-estrogen receptor alpha (ER; Millipore Corporation, Billerica, MA) or a 1:1000 dilution of rabbit anti-DOR1-(3–17). The sections were washed with three changes of PBS and coincubated with 3 µg/ml donkey anti-guinea pig IgG conjugated to Cy3 and with 3 µg/ml donkey anti-rabbit IgG conjugated to Cy2 (Jackson Immunoresearch Laboratories, West Grove, PA) in PBS diluent for 2 h at room temperature. The sections were washed in three changes of PBS, rinsed in H₂O, dehydrated in increasing concentrations of ethanol (50–100%), and cleared in xylene. The slides were mounted with coverslips using DPX Mountant (Fluka Chemical Corp., Ronkonkoma, NY).

The specificity of the antisera used in these studies was tested using absorption controls. Controls were performed by adding 10 µg of the peptide, against which the antiserum was raised to 1 ml of the diluted antiserum. In all cases, the resulting labeling was substantially reduced or abolished (Supplemental Figs. 1 and 2).

Images were collected using either a conventional fluorescence microscope (Olympus BX-50; Olympus, Tokyo, Japan) or a confocal microscope equipped with laser lines at 488 and 543 nm (Olympus Fluoview 1000). Confocal images were collected with a 60x, 1.4 NA, oil immersion objective. All images are of coronal sections. Digital images were adjusted for publication (e.g., sharpened, resized, merged, and adjusted for brightness and/or contrast) with Photoshop (Adobe, San Jose, CA) or Olympus Fluoview Viewer (Olympus). Control and experimental images were manipulated identically.

Quantitative immunocytochemical studies were performed using four HSP and four control rats; one to six sections per segment per rat were evaluated for these studies. The density of DOR-ir was evaluated using the ImageJ program. Images were collected using a 40x, 0.85 NA objective, filtered using a Fourier short-pass filter (10 pixel maximum), and thresholded at an intensity of 112, and the number of above-threshold pixels were counted. The numbers of terminals double-labeled for DOR-ir and DYN-ir were also counted. Because automated methods proved unreliable in this case, counting was performed by eye using pairs of images obtained with a 40x, 0.85 NA objective.

DYN-ir neurons, ER-ir neurons, and DYN/ER double-labeled neurons were counted by eye directly from the tissue using a 20x, 0.7 NA objective. Given that control and HSP tissue were mounted on the same block (see above), it was not possible for the tissue to be evaluated "blindly". To validate the objectivity of our observations, we used images taken from a subset of the data. Pairs of images were made of the left and right dorsal horns from the same spinal cord (in and adjacent to the lateral substantia gelatinosa); images from the right side were identified with regard to experimental group, whereas the identities of the images from the left sides were encoded. The highly significant correlation between the numbers of cells counted on the two sides (Pearson's r value = 0.5526; p = 0.0051; 24 pairs of images from L₆;ER-ir evaluated) is strong evidence showing that our awareness of the experimental conditions did not influence results.

Statistical comparisons were made using the Prism software package (GraphPad Software, Inc., San Diego, CA). Unpaired t-tests or Fisher's exact tests were employed.

	Results

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Distributions of DYN and DOR. Observations were made in spinal segments L₅ to S₂ of four control and four HSP rats. In control and HSP females, DOR-ir occurred almost entirely as fibers rather than cell somata (Fig. 1a). Labeling was most dense in the superficial dorsal horn and had a distribution similar to that of small-diameter primary afferent fibers. DOR-ir fibers were also found widely throughout the neck of the dorsal horn and the remainder of the spinal cord gray matter. These findings are consistent with previous descriptions of DOR-ir (Dado et al., 1993; Arvidsson et al., 1995a).

View larger version (77K): [in this window] [in a new window]   Fig. 1. DOR-ir (a) and DYN-ir (b) in the L6 spinal cord dorsal horns of control female rats. a, DOR-ir was observed in varicosities but generally not somata. Inset shows a high magnification view of region outlined by the box. b, DYN-ir was observed in both varicosities and somata. Inset shows DYN-ir somata in the superficial dorsal horn. The distribution of DOR-ir and DYN-ir agrees with previous reports. Bar = 100 µm for main images; 25 µm for insets.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Numbers of DYN-ir neurons observed in control female (open bars) and HSP rats (filled bars) at different spinal levels. Significantly more DYN-ir neurons were observed in L5 and L6 of HSP animals (*, p = 0.0122; **, p = 0.0042).

Coexpression of DYN and DOR. The regulation of DYN release by activation of DOR suggested that DORs might be expressed by DYN neurons. Consistent with previous observations (Dado et al., 1993; Arvidsson et al., 1995a), we did not observe DOR expression by somata. However, DYN-ir terminals frequently (Fig. 3), although not always (Fig. 4), expressed DOR-ir. Fibers expressing DOR and DYN were observed in the dorsal horn and intermediate gray matter of both HSP and control rats. We did not observe significant differences in either the numbers of double-labeled terminals in lamina V in L₅ to S₂ (p > 0.2, unpaired t-tests) or in the density of DOR-ir in this region (p > 0.34, unpaired t-tests). The absence of an increase in the number of terminals coexpressing DOR and DYN during HSP suggests that DORs are not present in the neurons in which increased DYN expression occurs. In addition to the colocalization of DYN and DOR, DOR-ir varicosities frequently apposed DYN somata in control and HSP rats (Fig. 4). This suggests that regulation of spinal DYN activity can occur via both direct and indirect consequences of DOR activation.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Expression of DOR-ir by DYN-ir terminals in control female (a–c) and HSP rats (d–f). a and d, DYN-ir. b and e, DOR-ir in the same microscopic fields as shown in a and d, respectively. c and f, merged images in which DYN-ir is red and DOR-ir is green. Arrowheads indicate double-labeling. Bar = 10 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 4. Apposition of DOR-ir terminals onto DYN-ir neurons in L6 of control female (a) and HSP rats (b). Red, DYN-ir; green, DOR-ir. DOR-ir terminals were frequently found in close proximity to DYN-ir neurons in the dorsal horn of both controls and HSP animals (arrowheads). Bar = 5 µm.

Terminal DORs Can Modulate DYN Release. Expression of DOR by DYN axon terminals would provide a mechanism by which activation of DORs could directly modulate DYN release and is consistent with a previous report from the Gintzler laboratory that DYN release can be modulated by DOR activation (Gupta et al., 2001); evoked release of DYN from minced spinal cord tissue can be dose-dependently inhibited by the DOR agonist DPDPE. However, the minced spinal cord preparation used in those studies contained DYN somata as well as DYN terminals, and release could have been modulated at either site. To determine whether an action of DPDPE on DORs expressed on terminals would be sufficient to inhibit DYN release, we investigated whether stimulated release of DYN from synaptosomes, which are devoid of somata, would still be inhibited by DPDPE. In the absence of DPDPE, basal release of DYN from spinal synaptosomes was 33.3 ± 2.3 pg per 6 min. The increment (stimulated minus basal) in the rate of DYN release evoked by 50 mM K⁺ was 11.1 pg per 6 min (Fig. 5, black bar). It is noteworthy that, in the presence of 1 µM DPDPE (following a 3-min pretreatment), the K⁺-evoked increase in the rate of DYN release was reduced to 6.3 pg per 6 min, a decrement of 4.8 pg (p = 0.03; n = 3; 43%; Fig. 5, open bar).

View larger version (13K): [in this window] [in a new window]   Fig. 5. Alteration by DPDPE of K+-evoked release of DYN from synaptosomes. The size of the increment evoked by 50 mM K+ was significantly smaller in the presence of DPDPE than in its absence (*, p = 0.03). Each column represents means ± S.E.M.

Coexpression of ER and DYN.

In dorsal horn of control female rats, we observed a moderate number of ER-ir structures that appeared to be neuronal nuclei and that were concentrated in the superficial dorsal horn and lateral reticulated area (lamina V) (Fig. 6). In HSP females, ER-ir cells were also found in the superficial dorsal horn and lamina V; in addition, they were now lightly distributed throughout the neck of the dorsal horn, the intermediate gray, and the ventral horn. The number of ER-ir nuclei was significantly higher (30%) in L₅, L₆, and S₁ of HSP animals (p < 0.02 in all cases; Fig. 7). No significant difference was observed in the S₂ segment (p > 0.4).

View larger version (84K): [in this window] [in a new window]   Fig. 6. Expression and coexpression of ER-ir and DYN-ir in L6 spinal cord dorsal horns of control female and HSP rats. a to c (left column), control female rats. d to f (right column), HSP rats. a and d, DYN-ir. b and e, ER-ir in the same microscopic fields as shown in a and d, respectively. Localization of ER-ir in structures labeled by Hoechst dye 33258 (inset in b) indicates its expression in cell nuclei. c and f, merged images showing the relationships between DYN-ir (red) and ER-ir (green). Note expression of ER-ir by some (arrows), but not all, DYN-ir neurons and increased coexpression in HSP animals. Line drawings in c and f show regions from which images were obtained. Bar in F = 25 µm and applies to a to f; bars in line drawings = 1 mm and apply only to those drawings.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Numbers of neurons expressing ER-ir in the dorsal horns of different spinal segments in control females (open bars) and HSP rats (filled bars). Significantly more ER-ir neurons were observed in HSP rats in L5, L6, and S1 (*, p = 0.0159; **, p = 0.0057; ***, p < 0.0001).

View larger version (18K): [in this window] [in a new window]   Fig. 8. Coexpression of ER-ir and DYN-ir in spinal cord dorsal horn. A, numbers of neurons coexpressing ER-ir and DYN-ir in the dorsal horns of different spinal segments in control females (open bars) and HSP rats (filled bars). Significantly more ER-ir neurons were observed in HSP rats in L6 and S1 (unpaired t test; *, p = 0.0261; ***, p < 0.0001). B, the proportion of DYN-ir neurons that coexpressed ER-ir in the dorsal horns of different spinal segments in control females (open bars) and HSP rats (filled bars). A significantly higher fraction of DYN-ir neurons were double-labeled for ER-ir in segment L6 in HSP rats (*, p = 0.0302; Fisher's exact test).

	Discussion

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GSA is a phenomenon that has been observed in humans as well as other species. Despite the fact that many of the contributing cell-biological substrates mediating it are fairly well understood, the relevant anatomical circuits are not. Current findings shed light on the anatomical organization of this system and allow three general conclusions to be drawn: 1) estrogen receptors (specifically, ER) are expressed by DYN-ir neurons; 2) DYN-ir varicose fibers in the spinal cord dorsal horn and intermediate gray matter also express DOR-ir; and 3) activation of DORs located on DYN varicosities is sufficient to inhibit K⁺-evoked DYN release.

Previous studies have revealed the critical importance of the enhanced activity of the spinal cord DYN/KOR analgesic pathway to both GSA and HSPA (Gupta et al., 2001). However, demonstration of enhanced release of spinal DYN during gestation and HSP and the consequent elevation of nociceptive response thresholds could not distinguish between direct and indirect effects of E₂/P on spinal DYNergic neurons. The current finding that ER is expressed by DYN neurons indicates that spinal DYNergic activity can be directly modulated by ERs.

During HSP, in L₆, there were significant increases in 1) the number of DYN-ir cells, 2) the number of ER-ir cells, 3) the number of cells double-labeled for DYN-ir and ER-ir, and 4) the proportion of DYN-ir cells coexpressing ER. The increase in the number of DYN-ir cells in L₆ (also seen in L₅) during HSP is consistent with the increment in DYN content of the lumbar spinal cord observed during gestation (Medina et al., 1993a,b) and suggests an expansion of DYNergic processing of afferent information (both uterine and cutaneous) during these conditions.

The mechanism responsible for the increased colocalization of ER and DYN cannot be discerned from current observations. Their increased coexpression could result from de novo expression of DYN by ER-bearing neurons. Alternatively, it is possible that ER expression is augmented among all populations of cells, including DYN neurons. Regardless, both mechanisms would have the same functional consequence of increasing the regulatory influence of E₂ on the function of spinal DYN neurons. The ability of physiological levels of E₂/P to augment ER expression during HSP is analogous to observations made in bone where the cellular content and activity of ER are regulated by E₂ (Lanyon et al., 2004; Zaman et al., 2006).

Previous studies by A.R.G. have found that afferent input from the uterus contributes to GSA; GSA is significantly reduced by sectioning of the hypogastric nerve (Gintzler et al., 1983), which carries afferent input from the rostral uterus to the upper lumbar spinal cord (L₁–L₃) (Berkley et al., 1993). L₆ and S₁ receive afferent input from the caudal uterus (including the cervix) via the pelvic nerve (Berkley et al., 1993). Thus, our present findings suggest either that the present observations are secondary to changes in the upper lumbar spinal cord or that the sectioning of the pelvic nerve may also affect GSA and HSPA.

The present studies employed profile counting rather than stereologically unbiased methods to quantify changes in immunohistochemical staining in spinal tissue of HSP rats, due to the difficulty using either the physical or optical dissector methods in the sections used in these studies. Profile counting is vulnerable to mis-estimation of cell numbers if the sizes of cells increase (Reed and Howard, 1998). However, it appears unlikely that this was the case given that there were no significant differences (either for DYN-ir or ER-ir) in the cross-sectional areas of cell profiles (for DYN) or nuclear profiles (for ER-ir) between control and HSP rats (p > 0.57 in both cases). Thus, it appears that the present results represent differences in cell numbers as well as differences in cell profiles.

Earlier studies revealed a variety of interactions between opioid systems and ERs. ER regulates synthesis and secretion of the DOR ligand methionine-enkephalin (Romano et al., 1988; Low et al., 1989; Amandusson et al., 1999). ER-mediated regulation of β-endorphin content and secretion is also well established (Lapchak, 1991; Nakano et al., 1991). Additionally, regulation by E₂ and ER of µ-opioid receptor density in the hypothalamus (Mateo et al., 1992; Joshi et al., 1993) and µ-opioid receptor internalization (Micevych et al., 2003) has been reported. The present demonstration of an association of ERs (ER) with DYNergic neurons in combination with the demonstrated influence of the estrous cycle on spinal KOR (Chang et al., 2000) emphasizes the influence of ovarian sex steroids on endogenous opioids.

Many of the actions of E₂ are mediated via protein kinase A (Gu and Moss, 1996; Auger et al., 2001; Mize and Alper, 2002). Furthermore, E₂ (via ER) modulates mitogen-activated protein kinase activation (Zhang et al., 2002), and phosphorylation (activation) of extracellular signal-regulated kinase 1/2 is induced by estradiol (Sétáló et al., 2002). We speculate that the enhanced release of spinal DYN noted during gestation and HSP (Gupta et al., 2001) could result from activation of ERs present on DYNergic neurons and the initiation of analogous signaling events. Moreover, membrane-bound ER can activate metabotropic glutamate receptors (Boulware et al., 2005). Thus, it is possible that estrogen via membrane ERs is able to directly affect the firing of DYN neurons.

The second notable finding is that DYN-ir fibers in the spinal cord dorsal horn and intermediate gray matter coexpress DOR-ir, suggesting that DOR agonists modulate DYN release by acting directly on DYN nerve terminals. A.R.G. and D.G. previously reported that evoked release of DYN from minced spinal cord tissue was dose-dependently inhibited by the DOR agonist DPDPE (Gupta et al., 2001). The minced spinal cord preparation used in those studies contained both DYN somata and DYN terminals, precluding differentiation between these two sites of action. However, somata are not present in the spinal cord synaptosomal preparation used in the current study. Thus, the most parsimonious explanation for ability of DPDPE to inhibit DYN release from spinal synaptosomes would be direct negative modulation of DYN release by terminal DORs. This confirms the functional relevance of our anatomical findings.

Expression of DOR by DYN terminals provides a mechanism by which DOR activation can modulate DYN release selectively at different synapses. The latter could allow pain to be modulated during pregnancy with a great deal of anatomical specificity. It is interesting that modulation of spinal DYN release by DOR reverses from inhibition to activation during HSP (Gupta et al., 2001), indicating its dependence on physiological state.

The present findings cannot directly explain some of the more striking characteristics of HSPA, i.e., the requirement for concomitant activation of both spinal DOR and KOR (Dawson-Basoa and Gintzler, 1998) and the shift from inhibition to enhancement of DOR modulation of spinal DYN release (Gupta et al., 2001). Nevertheless, the spatial relationships we observed among spinal DOR, ER, and the KOR agonist DYN justify the conclusion that they directly interact. We hypothesize that ER is expressed by neurons coexpressing DYN and DOR; the effects of ER activation on phosphorylation (see above) and other intracellular signaling events, e.g., altered DOR G protein coupling (Bao et al., 2003), could underlie the qualitative change in effects of DOR activation on DYN release. Coexpression of ER, DOR, and DYN would provide a means by which the effects of estrogens and DOR agonists on antinociceptive circuits could be integrated at DYN cells. This would enable DYN neurons to function as coincidence detectors for the local estrogen milieu and synaptic enkephalins.

Figure 9 integrates current findings and illustrates the circuitry that we propose underlies GSA and HSPA. Present results do not shed light on the extent to which the relationships among DYNergic neurons, DOR, and ER and their modulation by ovarian sex steroids are sexually dimorphic. This notwithstanding, demonstration in control animals of coexpression of DYN with DOR or ER suggests the relevance of these relationships to pain processing in cycling females. The antinociceptive utility of the release of spinal DYN is underscored by the recent demonstration of the dependence on spinal DYN release and KOR activation of intrathecal morphine antinociception in female rats (Liu et al., 2007). Thus, it is tempting to speculate that administration to the lumbosacral cord of appropriate regimens of ovarian sex steroids, alone or in combination with a DOR agonist, which would release DYN after treatment with ovarian sex steroids (Gupta et al., 2001), could represent a novel pharmacotherapeutic approach in treating chronic pelvic pain in women.

View larger version (27K): [in this window] [in a new window]   Fig. 9. Illustration of proposed spinal circuitry underlying the analgesic effects of ovarian sex steroids. Because these effects have been shown to be due to spinal release of DYN, we propose that the discomfort of pregnancy is ameliorated by DYN inhibition of nociceptive neurons in the dorsal horn, possibly including relay neurons such as spinothalamic tract neurons. A, we envision that relay neurons (RNs) receiving uterine or cutaneous afferents are inhibited by DYN neurons that express ER. Activation of ERs in pregnancy and HSP increases expression and release of DYN (Medina et al., 1993a,b). Release of DYN can inhibit nociceptive spinal neurons, including spinothalamic tract neurons (Willcockson et al., 1986), presumably via activation of KOR. B, detail of DYN neuronal contact. DYN terminals express DOR; activation of DOR during pregnancy or HSP promotes DYN release (Gupta et al., 2001). DRG, dorsal root ganglion.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.139816.

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, 17β-estradiol, estrogen; P, progesterone; KOR, -opioid receptor; NA, numerical aperture; DPDPE, D-Pen⁵-enkephalin; PBS, phosphate-buffered saline.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

¹ Current affiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky.

Address correspondence to: Dr. Alan Gintzler, Box 8, Department of Biochemistry, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203. E-mail: alan.gintzler{at}downstate.edu

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