Spinal and supraspinal N-methyl-D-aspartate and melanocortin-1 receptors contribute to a qualitative sex difference in morphine-induced hyperalgesia.

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-methyl-d-aspartate receptor (NMDAR) and melanocortin-1 receptor (MC1R) antagonists sex-dependently reverse MIH during morphine infusion (40mg/kg/24h) 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.

C-Fos activation in the periaqueductal gray following acute morphine-3ß-D-glucuronide or morphine administration.

Morphine-3ß-D-glucuronide (M3G), a primary morphine metabolite, evokes hyperalgesia in mice and rats and putatively mediates hyperalgesia associated with morphine (MOR) administration. However, M3G does not act via opioid receptors and its locus of activity in the CNS is unknown. Here we assessed the density of neurons immunoreactive for c-Fos, an immediate early gene regulated by neuronal activity, in the periaqueductal gray (PAG), a midbrain region critical to pain modulation, in male CD-1 mice after MOR and M3G exposure. Mice were injected with acute doses of MOR or M3G following a pre-injection of saline (SAL) or the opioid antagonist naltrexone (NTX), perfused 3 h later, and labeled for c-Fos using immunohistochemistry. Labeled image stacks taken from the PAG were then analyzed on a confocal microscope for the number of neurons showing c-Fos expression. Relative to controls, significant but similar increases in the mean density of PAG c-Fos immunoreactive neurons were observed in mice pre-injected with SAL then M3G or morphine. However, NTX pre-injection blocked this increase in MOR but not M3G injected mice. The data demonstrate for the first time a CNS locus for M3G activity. Consistent with previous observations, this M3G activity is not mediated by opioid receptors.

Progesterone rapidly recruits female-typical opioid-induced hyperalgesic mechanisms.

Continuous morphine treatment can paradoxically increase nociception (i.e. hyperalgesia) in male and female mice, but sex differences have been reported. Here, we studied progesterone modulation of these differences by assessing nociception on the tail-withdrawal test in male and female mice rendered hyperalgesic during continuous infusion of two different morphine doses (1.6 and 40.0mg/kg/24h). Although the lower morphine infusion dose increased nociception in both sexes by infusion Day 4, this hyperalgesia dissipated by Day 6 in males and ovariectomized females, but not gonadally intact females. A single subcutaneous progesterone (0.0016mg/kg) injection to males and ovariectomized females on Day 6 caused hyperalgesia to recur within 30min and to persist for a minimum of 120min. The larger morphine infusion dose also increased nociception in both sexes on Days 4 and 6. However, the NMDA receptor antagonist MK-801 (0.05mg/kg) reversed hyperalgesia in males and ovariectomized females but not gonadally intact females on infusion Day 6. Subcutaneous progesterone (0.0016mg/kg) injection inhibited this reversal in male and ovariectomized female mice but had no effect on nociception in saline-infused mice of either sex. These data confirm our previous findings that male and female mice utilize distinct hyperalgesic mechanisms, and show for the first time that a single progesterone bolus dose can recruit female-typical hyperalgesia in ovariectomized females and males.

The contribution of MOR-1 exons 1-4 to morphine and heroin analgesia and dependence.

Although morphine and heroin analgesia is mediated by mu-opioid receptors encoded by the MOR-1 gene, distinct isoforms are involved. Both opioids also induce dependence by acting at mu-opioid receptors, but which variants are utilized is not known. Here, we assayed morphine and heroin analgesia and dependence in mice treated with antisense oligodeoxynucleotides (AO) targeting MOR-1 exons 1-4. Whereas AOs targeting exons 1 and 4 blocked morphine analgesia, those targeting exons 2 and 3 blocked heroin analgesia. Neither morphine nor heroin analgesia was compromised 5 days after the last AO injection. In morphine and heroin dependent mice, only exon 1 AO significantly reduced jumping incidence during naloxone (50mg/kg) precipitated withdrawal. Neither analgesia nor withdrawal jumping was attenuated in controls pretreated with saline or a mismatch oligodeoxynucleotide control sequence. While these data confirm previous reports that morphine and heroin analgesia are not mediated by a single mu-opioid receptor, both opiates nonetheless apparently induce dependence via a mu-opioid receptor isoform containing exon 1. For heroin, the possibility that analgesia and dependence are mediated by distinct mu-opioid receptor isoforms offers the prospect of developing potent opiate analgesics possessing reduced dependence liability.

Sex differences in hyperalgesia during morphine infusion: effect of gonadectomy and estrogen treatment.

Morphine treatment can paradoxically increase nociception (i.e. hyperalgesia). Since there are putative sex differences in nociception and morphine sensitivity, we compared nociception in male and female mice using the tail-withdrawal test during continuous infusion of two morphine doses (1.6 and 40.0 mg/kg/24 h). Both doses caused hyperalgesia in both sexes, but onset in females always preceded that of males. Although the larger dose initially evoked analgesia, naltrexone (NTX) pellets implanted prior to morphine infusion abolished analgesia but not hyperalgesia. Distinct sex differences also characterized each morphine dose. Specifically, the lower morphine dose caused hyperalgesia that dissipated after 6 days in males but persisted in females for a minimum of 14 days. Despite this difference, N-methyl-d-aspartate (NMDA) receptor antagonists reversed hyperalgesia in both sexes. In contrast, the higher morphine dose evoked hyperalgesia that resolved concurrently in both sexes, but hyperalgesia was reversed by NMDA receptor antagonists in males only. Ovariectomy (OVX), but not OVX followed by estrogen treatment, abolished both sex differences, and resulted in females exhibiting the male-typical pattern. This study thus demonstrates NTX-insensitive morphine hyperalgesia in females as previously reported for males. However, females utilized hyperalgesic mechanisms which were distinct from those employed by males. Data from females subject to OVX/estrogen replacement further indicate that females possess functional male-typical hyperalgesic mechanisms, but are diverted from their use by ovarian sex steroids. Finally, the finding that each morphine infusion dose was characterized by a unique sex difference provides additional evidence for distinct multiple hyperalgesic systems.

A survey of acute and chronic heroin dependence in ten inbred mouse strains: evidence of genetic correlation with morphine dependence.

Heroin and morphine exposure can cause physical dependence, with symptoms manifesting during their withdrawal. Inter-individual differences in symptom frequency during morphine withdrawal are a common finding that, in rodents, is demonstrably attributable to genotype. However, it is not known whether inter-individual differences characterize heroin withdrawal, and whether such variation can be similarly influenced by genotype. Therefore, we injected mice of ten inbred strains with acute and chronic heroin doses and compared their jumping frequencies, a common index of withdrawal magnitude, during naloxone-precipitated withdrawal. The data revealed significant strain frequency differences (range after acute and chronic heroin injection: 0-104 and 0-142 jumps, respectively) and substantial heritability (h(2)=0.94 to 0.96), indicating that genetic variance is associated with heroin withdrawal. The rank order of strain sensitivity for acute and chronic heroin withdrawal jumping, and for the current heroin and previous morphine strain data, were significantly correlated (r=0.75-0.94), indicating their genetic and, ultimately, physiological commonality. These data suggest that the genetic liability to heroin dependence remains constant across a period of heroin intake, and that heroin and morphine dependence may benefit from common treatment strategies.

Acute and chronic heroin dependence in mice: contribution of opioid and excitatory amino acid receptors.

Opioid and excitatory amino acid receptors contribute to morphine dependence, but there are no studies of their role in heroin dependence. Thus, mice injected with acute or chronic heroin doses in the present study were pretreated with one of the following selective antagonists: 7-benzylidenenaltrexone (BNTX), naltriben (NTB), nor-binaltorphimine (nor-BNI; delta1, delta2, and kappa opioid receptors, respectively), MK-801, or LY293558 (NMDA and AMPA excitatory amino acid receptors, respectively). Naloxone-precipitated withdrawal jumping frequency, shown here to be a reliable index of heroin dependence magnitude, was reduced by BNTX or NTB in mice injected with both acute and chronic heroin doses. In contrast, nor-BNI did not alter jumping frequencies in mice injected with an acute heroin dose but significantly increased them in mice receiving chronic heroin injections. Continuous MK-801 or LY293558 infusion, but not injection, reduced jumping frequencies during withdrawal from acute heroin treatment. Their delivery by injection was nonetheless effective against chronic heroin dependence, suggesting mechanisms not simply attributable to NMDA or AMPA blockade. These data indicate that whereas delta1, delta2, NMDA, and AMPA receptors enable acute and chronic heroin dependence, kappa receptor activity limits the dependence liability of chronic heroin. With the exception of delta1 receptors, the apparent role of these receptors to heroin dependence is consistent with their contribution to morphine dependence, indicating that there is substantial physiological commonality underlying dependence to both heroin and morphine. The ability of kappa receptor blockade to differentially alter acute and chronic dependence supports previous assertions from studies with other opioids that acute and chronic opioid dependence are, at least in part, mechanistically distinct. Elucidating the substrates contributing to heroin dependence, and identifying their similarities and differences with those of other opioids such as morphine, may yield effective treatment strategies to the problem of heroin dependency.

Neonatal testosterone exposure influences neurochemistry of non-opioid swim stress-induced analgesia in adult mice.

The effects of neonatal hormone manipulations on swim stress-induced analgesia (SSIA) magnitude and neurochemical quality were examined in Swiss-Webster mice of both sexes. Previous research has indicated that non-opioid SSIA mechanisms in adult Swiss-Webster mice are sexually dimorphic. Male mice exhibit non-opioid SSIA following a 3-min swim in cold (15 degrees C) water that is antagonized by the non-competitive NMDA antagonist MK-801 (dizocilpine; 0.075 mg/kg), whereas female mice do not display NMDA-mediated analgesia in the presence of estrogen. Since male and female mice show equipotent magnitudes of SSIA, it was concluded that female mice display a neurochemically distinct, estrogen-dependent SSIA mechanism specific to their gender. In the present study, female mice exposed to testosterone during the neonatal period display NMDA-mediated analgesia even in the presence of estrogen in adulthood. Thus, expression of the female-specific, estrogen-dependent SSIA mechanism previously described may be dependent on the absence of testosterone during early ontogeny.

Sex differences in the antagonism of swim stress-induced analgesia: effects of gonadectomy and estrogen replacement.

Sex differences in the neurochemical mediation of swim stress-induced analgesia (SSIA) were examined in Swiss-Webster mice. Intact and gonadectomized adult mice of both sexes were tested for their analgesic response (hot-plate test) to 3 min of forced swimming in 15 degrees C and 20 degrees C water. SSIA resulting from 15 degrees C swim was previously shown to be naloxone-insensitive (i.e., non-opioid) whereas SSIA resulting from 20 degrees C swim produced an analgesia that was partially reversible by naloxone (i.e., mixed opioid/non-opioid). The non-opioid components of these SSIA paradigms were attenuated by the N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (MK-801). We now report that in males, but not females, dizocilpine (0.075 mg/kg, i.p.) and naloxone (10 mg/kg, i.p.) antagonized the non-opioid and opioid components of SSIA, respectively. After ovariectomy, females displayed a pattern of antagonism similar to males such that dizocilpine attenuated non-opioid SSIA, although naloxone remained ineffective in antagonizing 20 degrees C SSIA. Thus, SSIA in intact females was neither opioid- nor NMDA-mediated, yet it was of similar magnitude to the SSIA displayed by intact males. In separate experiments, estrogen replacement (estrogen benzoate; 5.0 micrograms/day, i.p.) administered to ovariectomized mice over a 6-8 day period reinstated the dizocilpine-insensitivity of 15 degrees C SSIA characteristic of intact females. However, a similar estrogen regimen administered to both intact and castrated males did not compromise the sensitivity to dizocilpine previously noted in male mice.(ABSTRACT TRUNCATED AT 250 WORDS)