Physiology & Behavior 147 (2015) 364–372

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Spinal and supraspinal N-methyl-D-aspartate and melanocortin-1 receptors contribute to a qualitative sex difference in morphine-induced hyperalgesia Caroline A. Arout a,⁎, Megan Caldwell b, Grace Rossi c, Benjamin Kest b,d,e a

Department of Psychiatry, Yale University School of Medicine, VA Connecticut Healthcare System, 950 Campbell Avenue, West Haven, CT 06516, United States Center for Developmental Neuroscience, The College of Staten Island, City University of New York, Staten Island, NY 10314, United States c Department of Psychology, Long Island University, Post Campus, Brookville, NY 11548, United States d Department of Psychology, The College of Staten Island, City University of New York, Staten Island, NY 10314, United States e Neuropsychology Doctoral Subprogram, The Graduate Center, City University of New York, New York, NY 10016, United States b

H I G H L I G H T S • Spinal & supraspinal MK-801 or MSG606 reverses MIH in males & females, respectively. • MSG606 was effective in reversing MIH after progesterone injection in OVX mice. • Spinal and supraspinal NMDARs & MC1Rs underlie a qualitative sex difference in MIH.

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Article history: Received 19 March 2015 Received in revised form 1 May 2015 Accepted 4 May 2015 Available online 14 May 2015 Keywords: Morphine Hyperalgesia Spinal Supraspinal NMDA receptor Melanocortin-1 receptor

a b s t r a c t Morphine elicits a paradoxical state of increased pain sensitivity, known as morphine-induced hyperalgesia (MIH), which complicates its clinical efficacy. We have previously shown that systemic injections of N-methylD-aspartate receptor (NMDAR) and melanocortin-1 receptor (MC1R) antagonists sex-dependently reverse MIH during morphine infusion (40 mg/kg/24 h) in male and female mice, respectively. This qualitative sex difference is ovarian hormone dependent, as NMDAR antagonists reverse MIH in ovariectomized females but are rendered ineffective following progesterone injection in OVX mice. Here, we utilized intrathecal and intracerebroventricular injection paradigms to assess the contribution of spinal and supraspinal receptors to this sex difference in male and female CD-1 mice. Specifically, we injected NMDAR and MC1R selective antagonists, MK-801 and MSG606 respectively, during morphine infusion. Results illustrated that both spinal and supraspinal MK-801 and MSG606 selectively reversed MIH in males and females, respectively, during morphine infusion. Furthermore, while MK-801 reversed MIH in ovariectomized (OVX) females, MSG606 was most effective in doing so in this same group following an acute subcutaneous progesterone injection. The present studies thus indicate that both spinal and supraspinal NMDARs and MC1Rs underlie the qualitative sex difference observed during morphine infusion in mice, and that the receptors in these loci are also sensitive to sex steroidal modulation. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Although morphine lies at the forefront of opioid treatment for moderate to severe pain, clinical treatment protocols are complicated by the paradoxical manifestation of hyperalgesia [1–4]. Whereas initial studies linked hyperalgesia to tolerance and withdrawal [5,6], more recent Abbreviations: NMDAR, N-methyl-D-aspartate receptor; MC1R, melanocortin-1 receptor; MIH, morphine-induced hyperalgesia; OVX, ovariectomy; i.c.v., intracerebroventricular; i.t., intrathecal; PAG, periaqueductal gray; RVM, rostroventral medulla; NTX, naltrexone. ⁎ Corresponding author at: Yale University School of Medicine, VA Connecticut Healthcare System, Department of Psychiatry, 950 Campbell Avenue, West Haven, CT 06516, United States. E-mail address: [email protected] (C.A. Arout).

http://dx.doi.org/10.1016/j.physbeh.2015.05.006 0031-9384/© 2015 Elsevier Inc. All rights reserved.

evidence suggests other mechanisms. Specifically, Juni et al. [7] demonstrated that morphine hyperalgesia can occur independently of prior or concurrent opioid receptor activity or analgesia, and is distinct from both withdrawal and tolerance. Morphine-induced hyperalgesia (MIH) is also subject to a qualitative sex-dependent interaction; of specific interest to which is the N-methyl-D-aspartate receptor (NMDAR) and the melanocortin-1 receptor (MC1R) [8–13]. Specifically, whereas hyperalgesia during systemic morphine infusion is reversed by an acute injection of the NMDAR antagonist MK-801 exclusively in male mice [8], female mice exhibit hyperalgesia of similar magnitude and duration that is reversed exclusively in this sex by the MC1R antagonist MSG606 [8,9]. Female mice do display NMDAR antagonist sensitivity; MK-801 reverses hyperalgesia in females after ovariectomy (OVX),

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but not when OVX is followed by estrogen or progesterone treatment [9, 10,14]. This finding indicates that, in addition to a distinct hyperalgesia system mediated by MC1Rs, female mice possess a male typical NMDAR hyperalgesic mechanism which is precluded from use by ovarian sex steroids. It is well documented that morphine modulation of nociception is regulated by multiple sites, notably including spinal loci such as superficial layers of the dorsal horn, dorsal root ganglia [15,16], and supraspinal loci such as the periaqueductal gray (PAG), and the rostroventral medulla (RVM) [15,17–22]. Furthermore, previous studies have also suggested that a multitude of these areas involved in morphine antinociception are also implicated in MIH. Specifically relevant to opioid-induced nociception are the dorsal horn, and reciprocal connections between the PAG and RVM [15,17,23–30]. However, the contribution of spinal and supraspinal loci to the qualitative sex differences characterizing MIH remains unknown. That is, the qualitative sex difference in the ability of NMDAR antagonists to reverse MIH was described after MK-801 was given systemically. Thus, presuming even distribution of the drug, the qualitative sex difference observed upon nociceptive testing 30– 90 min later [8,9] could have been mediated by either or both spinal and supraspinal loci. Although the contribution of MC1Rs to MIH was assessed by injecting MSG606 intracerebroventricularly in male and female mice, thus implicating supraspinal MC1Rs in female but not male MIH, the possible contribution of spinal MC1Rs is unknown. Given the substantial differences in protocols between our studies and those previously describing the spinal and supraspinal contributions to MIH, extrapolations between them are done tenuously at best. Such critical differences include, in our model, the manifestation of hyperalgesia in the absence of prior or concurrent opioid receptor activity and analgesia and, critically, the characterization that MIH is subject to qualitative sex differences. The goal of the present studies was to investigate the possible independent contribution of spinal and supraspinal mechanisms underlying qualitative sex differences in the model of MIH described above [7–10, 31,32]. Accordingly, male and female mice rendered hyperalgesic by continuous morphine infusion were tested for nociceptive sensitivity before and after receiving an MK-801 or MSG606 dose using the intracerebroventricular (i.c.v.) or intrathecal (i.t.) injection route of administration. Opioid receptor activity was concurrently blocked during the duration of the study by implanting mice with pellets containing the general opioid receptor antagonist naltrexone (NTX). In a final series of studies, we investigated whether spinal and supraspinal loci also contribute to male-typical NMDAR-mediated hyperalgesia in females when the qualitative sex difference is abolished following OVX and, finally, when the female typical pattern of MC1R-mediated hyperalgesia in OVX females is reinstated following progesterone. 2. Methods 2.1. Subjects Adult male and female CD-1 mice (Charles Rivers, Kingston, NY), six to ten weeks of age, were housed in cages of four with same-sex littermates. All mice were maintained on a 12:12-h light/dark cycle in a climate-controlled room (22 °C ± 2 °C) with ad lib access to food and filtered tap water. Following randomization into experimental or control groups, each subject was used once. For all conditions, n ≥ 8. 2.2. Nociceptive assay The warm-water tail-withdrawal test was chosen to assess nociception. This measure has repeatedly been shown to be stable and reliable in the context of recurrent testing [47,48,44]. Each subject was tested prior to NTX pellet implantation as a baseline measure, and before pump implantation to confirm no effect of NTX. All subjects were tested each day following pump implantation to assess hyperalgesia.

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On every testing day, each mouse was tested 15, 30, 45, and 60 min post-injection to assess any time course of hyperalgesia reversal. 2.3. Drugs and drug delivery protocols The current studies used morphine infusion pumps in mice concurrently treated with NTX pellets (30 mg). This protocol allowed for investigation of the mechanisms involved in MIH not confounded by initial or concurrent states of analgesia, and, as per previous protocols [7–9,31], eliminates the possibility of opioid receptor involvement. Furthermore, although MIH has been demonstrated in chronic paradigms that employ repeated injection [33,34] it is believed that in the context of discontinuous morphine delivery (i.e. multiple injections) hyperalgesia may be intensified by “mini withdrawal” episodes [1,35]. As opioid withdrawalinduced hyperalgesia has been shown to be mechanistically distinct from the opioid-induced hyperalgesia currently under investigation [36, 37], the use of osmotic pumps for morphine infusion eliminates the possible occurrence of withdrawal by providing uninterrupted, continuous morphine exposure over several days. Further, this is a commonly used and reliable method for sustained morphine delivery [7,9,10,31,32,38]. Twenty-four hours prior to initiation of morphine administration (day −1), NTX pellets were wrapped in nylon mesh and implanted subcutaneously in the nape of the neck. In rats, identical NTX pellets have been reported to substantially elevate NTX plasma levels 1 h after implant, and sustain pharmacologically active levels of NTX such that there is a greater than 50-fold rightward shift in the morphine analgesia dose response curve eight days later [39]. In mice, these pellets completely abolished the analgesic effect of an acute 10 mg/kg morphine injection starting 24 h after implant, coinciding here with the start of morphine infusion, and for a minimum of seven days thereafter [7]. One day after NTX pellet implantation (day 0), mice were implanted with osmotic pumps (Alzet Model 2001, Alza, Mountain View, CA) containing morphine (40 mg/kg/24 h, gift of NIDA) via a small dorsal midline incision made under anesthesia. All incisions were closed with stainless steel surgical staples. Where reported that, the non-competitive NMDAR antagonist, MK801 (Sigma-Aldrich, St. Louis, MO), and selective MC1R antagonist, MSG606, were injected via i.c.v. or i.t. route in a volume of 5 μl or 2 μl volume, respectively. MK-801 was dissolved in 0.9% physiological saline, while MSG606 utilized a 90% saline/10% DMSO vehicle. MSG606 (Cyclo-[(CH2)3CO-Gly-His-DPhe-Arg-D-Trp-Cys(S-)]-Asp-Arg-Phe-GlyNH2) is a potent and novel cyclic thioether peptide, and selectively antagonizes the MC1R subunit. For a complete explanation of the synthesis of this compound, please see Juni et al. [8]. Controls for both compounds (saline or DMSO + saline for MK-801 or MSG606, respectively) were included to ensure no effect of the vehicle solution. In a subset of subjects, an acute subcutaneous injection of progesterone (0.0016 mg/kg) was administered. Progesterone was obtained commercially (Sigma-Aldrich, St. Louis, MO), and dissolved in a sesame oil vehicle. The progesterone dose utilized in the current studies has been shown in mice to simulate characteristic physiological concentrations (per unit blood volume) of progesterone during the proestrous phase [40,41], and has been found to restore female-typical analgesic and hyperalgesic sensitivity after ovariectomy [10,14]. 2.4. Acute injection and testing protocols Mice subject to morphine infusion were rendered hyperalgesic by day 4, as assessed via the tail withdrawal test. Immediately after testing, all mice received either an i.c.v. or i.t. injection of MSG606 (1.5 mg/ml), an MSG606 vehicle (DMSO + saline), MK-801 (0.005 mg/ml), or an MK-801 vehicle (saline) performed under oxygen/isoflurane inhalant anesthesia. I.c.v. injections (5 μl volume) were made directly through the skull at a point 2 mm rostral and lateral to lambda at a depth of 3 mm using a 10 μl Hamilton micro-syringe fitted with a 27-gauge needle. A stainless steel wound clip was used to close the incision after each

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injection [42]. I.t. injections (2 μl volume) were performed under light oxygen/isoflurane inhalant anesthesia and administered by lumbar puncture, as adapted from Hylden and Wilcox [43]. Motor function (i.e. “step test” and performance on a 90° inclined grid) and righting reflexes were assayed in small (n = 6) separate groups of morphine infused male and female CD-1 mice injected with MSG606 at postinjection intervals corresponding to this drug's maximal effect on nociception. The dose of MK-801 used here has already been reported to have no impact on motoric functioning [7,9,44] and was thus not additionally assayed here. In order to address the possibility that antagonists exerted their effects following caudal or rostral spread in i.c.v. or i.t. injection paradigms, respectively, we performed confirmatory studies under both injection conditions. Specifically, an injection of 0.5% methylene blue solution was injected either via i.c.v. or i.t. routes of administration, and animals were sacrificed either immediately following, or 30 min postinjection. At either point, brains or spinal cords were extracted and dissected. Following i.c.v. injection, we observed no caudal spread of the injection volume to the spinal cord. In the i.t. paradigm, the dispersal of the 2 μl-injection volume used in these studies was observed to travel approximately 0.5 to 1 cm within 30 min. Further, a study utilizing a similar i.t. protocol to ours confirmed our observations after injecting a 5 μl volume of 1% methylene blue solution, mirroring our findings of a rostral and caudal dispersal of 0.5 cm [45]. Nociception was assessed via the tail withdrawal test in all mice, every 15 min for 60 min post-injection. A modified version of the tailwithdrawal test [46] was chosen for its stability in the context of repeated testing [44,47,48]. Studies described here were performed with a water temperature of 47.5 °C; in pilot studies utilizing this temperature, baseline latencies of 9–10 s were consistently obtained, thus minimizing the possibility of floor effects during hyperalgesia. Nociception was tested near mid-photophase to reduce possible circadian effects on nociception [49]. Nociception was always assessed prior to any surgical procedure. Animals with tails that were visibly injured or otherwise deformed or diseased were excluded from the study, given their likely effect upon peripheral pain processing in the tail. Likewise, animals that showed obvious signs of disease, motor impairment, or failed to exhibit the tail withdrawal response during baseline behavioral assessments (withdrawal latencies exceeding 30 s), were not included for further analysis. Finally, animals that demonstrated motor impairments following i.c.v. or i.t. injections were not included in final data analyses. 2.5. Ovariectomy Where indicated, female mice were subjected to ovariectomy (OVX) surgery through a single ventral midline incision. The fallopian tubes were then exposed and ligated with surgical silk proximal to the ovaries before removing the distal ends (including the ovaries and surrounding ovarian fat). Complete ovarian removal was visually confirmed by a second independent investigator. Incisions were then closed using surgical silk sutures (3-0) and mice were allowed a 20-day recovery period before undergoing any testing manipulations. This surgical procedure is quite common and demonstrated to be an effective way to disrupt estrus cycles and effectively prevent estrogen expression and subsequent circulation [50]. Following recovery, NTX pellet and morphine pump administration protocols were followed in these OVX females, as well as intact males. On day 4, mice of both sexes were rendered hyperalgesic. In order to assess abolition of female-typical mechanisms following OVX, and subsequent recruitment of male-typical systems in these OVX females, acute i.c.v. or i.t. injections of MSG606, MSG606 vehicle, MK-801, or MK-801 vehicle were made. On day 6, both sexes remained hyperalgesic. To investigate the possibility that female-typical mechanisms are recruited due to ovarian hormones, identical i.c.v. or i.t. injection procedures were followed, preceded by a subcutaneous injection of progesterone (0.0016 mg/kg) 30 min earlier in both OVX females and intact males.

Nociception was assessed via the tail withdrawal test in all mice, every 15 min for 60 min post-injection. 2.6. Data analysis Opioid-induced hyperalgesia was expressed as raw withdrawal latencies, which were analyzed using three-way analyses of variance (sex ∗ antagonist drug ∗ time) followed by a Fisher's LSD (protected ttest) for post-hoc comparisons. Data from i.c.v. and i.t. injection paradigms were analyzed separately. An alpha value of b 0.05 indicated significant differences. 3. Results Latencies on the tail withdrawal test were significantly reduced as compared to baseline in all subjects by day 4 of continuous morphine administration, indicative of hyperalgesia (p b 0.05; Figs. 1–4). No increase in tail flick latency was observed in either sex following injection of either control under any circumstances (p N 0.05). Results for each condition are discussed below. 3.1. Supraspinal hyperalgesia during morphine infusion Following morphine infusion, an i.c.v. injection of MK-801 resulted in a significant increase of tail withdrawal latency in exclusively males 15 min post-injection, an effect which was strongest at 15 min postinjection as illustrated by a 4.3 s increase in latency, and persisted through the 60 min time point (p b 0.05; Fig. 1B). In contrast, female latencies significantly increased in a magnitude equal to that observed in males, 15 min after injection of the MC1R antagonist MSG606 but not MK-801 (p b 0.05; Fig. 1A). This effect persisted for 60 min postinjection, and was best-illustrated at 15 min post-injection, by a 5 s increase in average tail withdrawal latency. 3.2. Spinal hyperalgesia during morphine infusion Mirroring the results of the aforementioned i.c.v. studies, decreased tail withdrawal latencies following morphine infusion were reversed in males exclusively by a single i.t. injection of MK-801 at 15 min postinjection, an effect which resolved within the 60 min testing period (p b 0.05; Fig. 2B). This effect was strongest at 30 min post-injection, illustrated by a 3.2 s increase from baseline tail withdrawal. In contrast to males, an acute i.t. injection of MSG606, but not MK-801, significantly increased tail-flick latencies in females from 6.3 s at baseline, to 8.5 s at the most efficacious point (30 min) (p b 0.05; Fig. 2A). 3.3. Supraspinal hyperalgesia in OVX females and intact males during morphine infusion Following i.c.v. injection of MK-801, a significant increase in tail withdrawal latencies was observed in both intact males and OVX females at 15 min post-injection on day 4 (p b 0.05; Fig. 3). No effect was observed in either sex following injection of MSG606 (p N 0.05; Fig. 3). Interestingly, on day 6, an acute subcutaneous progesterone injection induced a short-lived effect in males, as both i.c.v. injections of MK-801 and MSG606 caused a significant increase in tail withdrawal latency that resolved within the 60-minute post-injection testing period (p b 0.05; Fig. 3B). Similarly, progesterone replacement on day 6 in OVX females allowed the recruitment of both the NMDAR and the MC1R system. However, it is notable that reversal by MK-801 was significant only at the 15-minute and 60-minute time-points in OVX females following acute progesterone, and was marked by a meager 1.2 s and 1.3 s increase in tail withdrawal latency, respectively (p b 0.05; Fig. 3A).

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Fig. 1. Time course of hyperalgesia in CD-1 females (A) and males (B) receiving continuous morphine infusion, as assessed by the tail-withdrawal test. Mice were implanted with NTX pellets (30 mg) on day −1, and implanted with osmotic pumps containing morphine (40 mg/kg/24 h) on day 0. When hyperalgesic (day 4), mice were administered acute i.c.v. injections of one of the following: the NMDA receptor antagonist MK-801 (.05 mg/kg), the MC1R antagonist MSG606 (1.5 mg/ml), the MK-801 vehicle, or the MSG606 vehicle. Following, all mice were assayed for nociception every 15 min for 60 min. Values represent mean tail-withdrawal latency ± S.E.M.; following antagonist injections at “0”, significant increases in withdrawal latencies relative to baseline values obtained on day 4 are indicated (+), denoting significant reversal of hyperalgesia assessed at 15, 30, 45, and 60 min post-injection. Where significant, hyperalgesia is also indicated (−).

3.4. Spinal hyperalgesia in OVX females and intact males during morphine infusion On day 4, a significant increase in tail flick latency was observed after i.t. injection of exclusively MK-801 in both intact males and OVX females at 15 min post-injection, illustrated by an average 4.3 s increase in latency (p b 0.05; Fig. 4). In contrast to the aforementioned i.c.v. studies, progesterone on day 6 had no effect on males (p N 0.05), as exclusively i.t. MK-801 mediated increases in tail-flick latency over the course of testing (p b 0.05; Fig. 4B). However, progesterone replacement on day 6 in OVX female mice mediated spinal NMDAR and MC1R increases in tail-flick latencies (p b 0.05; Fig. 4A). 4. Discussion The overall aim of the current series of studies was to elucidate the precise location(s) of action of the regulatory mechanisms that underlie MIH. The current studies demonstrate several findings that expand upon what is currently known about this phenomenon: 1) MIH elicited by continuous infusion is reversed by i.c.v. administration of MK-801 in

exclusively males; this same state of MIH is reversed exclusively by i.c.v. administration of the MC1R antagonist MSG606 in females only; 2) MIH is reversed by i.t. administration of MK-801 in exclusively males, while MIH is significantly reduced by i.t. administration of the MC1R antagonist MSG606 in females only; 3) Following OVX, females exhibit male-typical patterns of hyperalgesia following continuous morphine administration; 4) After an acute injection of systemic progesterone, female typical patterns of hyperalgesia are restored following i.c.v. antagonist administration; and 5) Following an acute injection of systemic progesterone, there appears a spinal MC1R mechanism that modulates female-typical MIH; 6) Following progesterone administration, males are capable of recruiting supraspinal female-typical hyperalgesic mechanisms. These findings are discussed in detail below. 4.1. Morphine-induced hyperalgesia is reversed by dose- and sexdependent mechanisms specific to supraspinal loci Systemically administered opioids produce profound antinociceptive effects peripherally, supraspinally, and spinally, but the precise

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Fig. 2. Time course of hyperalgesia in CD-1 females (A) and males (B) receiving continuous morphine infusion, as assessed by the tail-withdrawal test. Mice were implanted with NTX pellets (30 mg) on day −1, and implanted with osmotic pumps containing morphine (40 mg/kg/24 h) on day 0. When hyperalgesic (day 4), mice were administered acute i.t. injections of one of the following: the NMDA receptor antagonist MK-801 (.05 mg/kg), the MC1R antagonist MSG606 (1.5 mg/ml), the MK-801 vehicle, or the MSG606 vehicle. Following, all mice were assayed for nociception every 15 min for 60 min. Values represent mean tail-withdrawal latency ± S.E.M.; following antagonist injections at “0”, significant increases in withdrawal latencies relative to baseline values obtained on day 4 are indicated (+), denoting significant reversal of hyperalgesia assessed at 15, 30, 45, and 60 min post-injection. Where significant, hyperalgesia is also indicated (−).

mechanism of action of the ensuing hyperalgesia is not as well understood [8–10,51]. Our previous findings have shown there to be a systemically initiated sex- and dose-dependent mediation of MIH, such that acute bolus injections of MK-801 or MSG606 reversed this state differentially in male and female mice [8,9]. However, as our previous studies utilized systemic antagonist administration that allowed for widespread distribution throughout the peripheral and central nervous systems, it was unknown if the resulting reversal of hyperalgesia was modulated exclusively by supraspinal, spinal, or some combination of both central areas. The first series of current studies illustrates that hyperalgesia resulting from morphine infusion is reversed exclusively in males by the NMDAR antagonist MK-801, but reversed exclusively in females by i.c.v. injection of the MC1R antagonist MSG606. These outcomes suggest that these hormone-dependent mechanisms are in fact regulated by supraspinal loci that function independently of ascending nociceptive input. Noted, however, is the fact that both past and current findings are limited to high doses of morphine and that similar findings are not yielded by low morphine infusion doses [9], suggesting minimally two distinct dose-dependent supraspinal neural networks in the modulation of MIH.

4.2. Morphine-induced hyperalgesia is reversed by dose- and sexdependent mechanisms specific to spinal loci In order to better classify the location of the entirety of mechanisms modulating continuous MIH, we also investigated an i.t. receptor antagonist injection paradigm. These studies using spinal administration report that hyperalgesia resulting from continuous morphine infusion is reversed exclusively in males by the NMDAR antagonist MK-801, while significant reversal of MIH is seen in exclusively females following i.t. administration of the MC1R antagonist MSG606. The results of the current studies suggest that the hormonedependent mechanisms of MIH are regulated independently in the brain and spinal cord. That is, it appears that spinally regulated NMDAR antagonism remains sufficient for male mice in the reversal of this opioid-induced pronociceptive state. Conversely, female mice undergo significant hyperalgesic reversal by spinally administered MSG606 following continuous administration of high morphine doses. Thus, we report that in addition to a hyperalgesic neural network in the brain, there exists a spinal locus that independently regulates MIH. While the NMDAR mechanism that regulates hyperalgesia appears to be present and undoubtedly functional in the spinal cord, the current

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Fig. 3. Time course of hyperalgesia in OVX CD-1 females (A) and intact males (B) receiving continuous morphine infusion, as assessed by the tail-withdrawal test. Mice were implanted with NTX pellets (30 mg) on day −1, and implanted with osmotic pumps containing morphine (40 mg/kg/24 h) on day 0. When hyperalgesic (day 4), mice were administered acute i.c.v. injections of one of the following: the NMDA receptor antagonist MK-801 (.05 mg/kg), the MC1R antagonist MSG606 (1.5 mg/ml), the MSG606 vehicle, or the MK-801 vehicle. Following, all mice were assayed for nociception every 15 min for 60 min. By day 6, hyperalgesia was reinstated in all subjects. Following a baseline measure on this day (0), all subjects received an acute s.c. injection of progesterone (0.0016 mg/kg), followed by an acute i.c.v. injection 30 min later of MSG606, MK-801, or their respective vehicle controls. All mice were tested every 15 min post-antagonist injection for 60 min. Values represent mean tail-withdrawal latency ± S.E.M.; following antagonist injections at “0”, significant increases in withdrawal latencies relative to baseline values obtained on day 4 and day 6 are indicated (+), denoting significant reversal of hyperalgesia assessed at 15, 30, 45, and 60 min post-injection. Where significant, hyperalgesia is also indicated (−).

evidence suggests that the female-typical MC1R system also appears to be present in functionally significant concentrations beyond supraspinal loci. This provides contradictory evidence for reports that MC1Rs are found in dense concentrations in the PAG, and are not widespread in the spinal cord [52,53]. The current studies suggest that MC1Rs may be present in functionally significant concentrations in the spinal cord as well as the brain. However, further research is needed to corroborate this speculation. 4.3. Female hormones play a mediating role in morphine-induced hyperalgesia Although intact females typically utilize the MC1R system to mediate hyperalgesia, removal of circulating ovarian hormones via OVX should and did cause females to “switch” systems on day 4, where they utilized exclusively the NMDAR system to mediate MIH. However, subsequent progesterone replacement on day 6 caused females to resort back to their female-typical patterns, utilizing the MC1R system to mediate their hyperalgesia. Interestingly, females did appear to maintain use of the NMDAR system as well, although on what appears to be a weaker scale than the MC1R system. Further, an acute progesterone injection caused the additional recruitment of a female-typical system in males as well. Specifically, intact males were able to use either a

supraspinal NMDAR or MC1R system to mediate MIH. However, reversal by the NMDAR system was significantly stronger than reversal by the MC1R system in males. It is possible that a higher dose of progesterone may have had a more significant effect in both sexes lacking ovarian hormones. Nonetheless, it appears that not only are circulating ovarian hormones responsible for the switch between the NMDAR and MC1R system, but that these mechanisms work independently in both supraspinal and spinal loci. It is interesting that in the current studies, both male and OVX female mice recruited powerful supraspinal-level NMDAR systems during infusion of morphine. Further, progesterone caused both cohorts to recruit powerful NMDAR and MC1R supraspinal systems to mediate hyperalgesia. However, following progesterone replacement, spinally administered MSG606 has no effect on males, yet evokes a significant effect in females. Thus, there does appear to be a functional hyperalgesic mechanism in the spinal cord that involves the MC1R system. However, why males only recruit a supraspinal MC1R system after progesterone injection and not a spinal mechanism as well is unknown. 4.4. Limitations There are several limitations to the current studies. All assessments were conducted using morphine, a substance that preferentially binds

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Fig. 4. Time course of hyperalgesia in OVX CD-1 females (A) and intact males (B) receiving continuous morphine infusion, as assessed by the tail-withdrawal test. Mice were implanted with NTX pellets (30 mg) on day −1, and implanted with osmotic pumps containing morphine (40 mg/kg/24 h) on day 0. When hyperalgesic (day 4), mice were administered acute i.t. injections of one of the following: the NMDA receptor antagonist MK-801 (.05 mg/kg), the MC1R antagonist MSG606 (1.5 mg/ml), the MK-801 vehicle, or the MSG606 vehicle. After which, all mice were assayed for nociception every 15 min for 60 min. By day 6, hyperalgesia was reinstated in all subjects. Following a baseline measure on this day (0), all subjects received an acute s.c. injection of progesterone (0.0016 mg/kg), and an acute i.t. injection 30 min later of either MSG606, MK-801, MSG606 vehicle, or the MK-801 vehicle. All mice were tested every 15 min post-antagonist injection for 1 h. Values represent mean tail-withdrawal latency ± S.E.M.; following antagonist injections at “0”, significant increases in withdrawal latencies relative to baseline values obtained on day 4 and day 6 are indicated (+), denoting significant reversal of hyperalgesia assessed at 15, 30, 45, and 60 min post-injection. Where significant, hyperalgesia is also indicated (−).

to the μ opioid receptor, a characteristic common to virtually all opioids reported to cause hyperalgesia in humans and rodents [54]. While the current studies provide evidence for how these opioids cause hyperalgesia independently of opioid receptor activity, these findings are novel and thus we cannot say with utmost certainty if these assumptions can be extrapolated to include delta and kappa receptor opioids, or even other μ-preferring opioids administered under other paradigms [55]. Further studies that assess the hyperalgesic tendencies of different opioids are required before such comparisons can be made. Furthermore, since the dependent nociceptive measure in all studies described here, the tail withdrawal test, is a measure of thermal reflexive pain, it is possible that different results would be obtained on other nociceptive measures such as mechanical or chemical pain [55–57]. Further, the phase of the estrous cycle was not evaluated in female mice; thus, it is possible that animals were tested at varying stages of the estrous cycle. However, a study evaluating the estrous cycle's impact on sex differences in nociception reported negligible effects in mice [58]. Finally, only outbred CD-1 mice were used as subjects; different results may be found in mice of altered genetic background [56,57,59]. Thus, applicability beyond the narrow conditions described above should not be assumed.

5. Conclusions While a comprehensive understanding of the mechanisms underlying MIH still remains at large, the current series of studies have contributed several important findings in terms of sex differences in morphine-induced hyperalgesia. As suggested by its susceptibility to NMDAR antagonism, characteristic MIH in males (but not intact females) is under the influence of exclusively NMDARs. Thus, it is practical that others report greater increases in morphine analgesia in male mice following MK-801 administration relative to females [60]. Moreover, the failure of MK-801 to reverse hyperalgesia in females, and the ability for i.c.v. MSG606 to reverse male hyperalgesia following progesterone administration provide additional support favoring the existence of supraspinal sex-specific hyperalgesic mechanisms. Interestingly, the current studies demonstrate that if males are given female hormones, they have the ability to recruit an exclusively supraspinal femaletypical hyperalgesic mechanism. Conversely, males do not appear to recruit a spinal MC1R system after progesterone administration. It appears as though in such a case, the spinal NMDAR is superior. A supraspinal MC1R system is feasible due to this receptor's distribution in the PAG and in brain-glial cells (which more recently are speculated to play a role in hyperalgesia); however, this receptor is not

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reported to have widespread spinal distribution [13,53]. Interestingly, a recent study reported the involvement of MC4R, a subtype diffusely distributed both supraspinally and spinally, in neuropathic pain. Specifically, Delaney et al. found an upregulation of the fourth melanocortin subtype specifically in the spinal cord in response to peripheral nerve injury [52]. While this type of pain sensitivity is not identical to MIH, these findings suggest the involvement of more than one differentially distributed melanocortin receptor subtype in the modulation of female-specific pain. While the current studies further define MIH during continuous infusion, the mechanisms underlying this paradoxical state still remain unclear. In particular, we suggest a more complicated sex-dependent mechanism, in that males may also recruit female-typical systems while still producing their own gonadal hormones. Interestingly, both male castration and the administration of gonadal hormones in OVX females decrease the potency of morphine-induced analgesia, suggesting that male hormones increase the efficacy of opioid analgesics [61]. Quite problematic is the fact that, though there are vast amounts of animal studies that investigate pain, most paradigms use exclusively males in their studies. Ironically, females are often excluded due to the very issue that causes their sex-differentiated pain: fluctuations in ovarian hormones. Because of their exclusion, this leaves the important area of female-typical pain unaddressed. For instance, the phase of the estrous cycle appears to play a role in animal models of female-mediated analgesia; specifically, systemically initiated analgesia is most potent during the metestrus and proestrus phases, and least effective during the estrous phase [55,61,62]. In humans, females tend to have higher pain tolerance during the follicular phase [55]. Additionally, preclinical literature suggesting superior analgesia in women following treatment with κ-agonists extrapolates to the clinical population. Thus, pharmacokinetic and pharmacodynamic sex differences are likely also at play, and it is likely that these hormone fluctuations play a role in hyperalgesia. While these findings by no means advocate for a reduction in the use of opioids in clinical settings, our findings shed a brighter light on sex differences in pain processing. Although there is an overwhelming amount of research on sex differences in pain, the clinical impact remains inadequate. As chronic pain is an increasingly growing issue that overwhelmingly encompasses the female patient, it is imperative that these sex differences are comprehensively understood in order to provide adequate care.

Acknowledgments This work was completed as part of C. A. Arout's doctoral dissertation, submitted to the Graduate Center of the City University of New York. The manuscript was prepared for publication during C. A. Arout's NIDA T32 Postdoctoral Fellowship at Yale University School of Medicine.

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