Aaron Juni

Naloxone-precipitated withdrawal jumping in 11 inbred mouse strains: evidence for common genetic mechanisms in acute and chronic morphine physical dependence

Physical dependence is a widely known consequence of morphine intake. Although commonly associated with prolonged or repeated morphine administration, withdrawal symptoms can be elicited even after a single prior morphine exposure. What remains contentious is the extent to which physical dependence following acute and chronic morphine treatment is mediated by common physiological substrates and, accordingly, represent distinct syndromes. The genetic relationship between acute and chronic morphine dependence was thus presently studied by comparing mice of 11 inbred strains (129P3, A, AKR, BALB/c, C3H/He, C57BL/6, CBA, DBA/2, LP, SJL, and SWR) for naloxone-precipitated withdrawal jumping responses using three subcutaneous morphine administration paradigms: acute (single injection) or chronic (three daily morphine injections for 4 days) injection, or chronic infusion (7 days via implanted osmotic minipumps). Although there were differences in the magnitude of withdrawal jumping between the three different morphine administration paradigms, large and significant strain differences were observed for each. In addition, the same strains were unusually sensitive or, conversely, altogether refractory to withdrawal jumping across all morphine treatment conditions. Overall, strain jumping means between acute and chronic dependence paradigms displayed a high degree of genetic correlation (r=0.87-0.95). The significant correlation between chronic morphine injection and continuous morphine infusion discounts the possible confounding effect of contextual learning and spontaneous withdrawal between chronic injections on the assessment of naloxone-precipitated withdrawal. Substantial heritability was also observed for acute and both paradigms of chronic dependence, with estimates ranging from h(2)=0.53 to 0.70. The present demonstration of a strong genetic correlation between physical dependence to morphine following acute and chronic treatment implies that genes associated with variable sensitivity in the two traits are the same, and is suggestive of shared physiological substrates. The data also demonstrate that the differential genetic liability to morphine physical dependence begins with, and is predicted by, the first morphine exposure.

Nociception increases during opioid infusion in opioid receptor triple knock-out mice

Opioids are extensively used analgesics yet can paradoxically increase pain sensitivity in humans and rodents. This hyperalgesia is extensively conceptualized to be a consequence of opioid receptor activity, perhaps providing an adaptive response to analgesia, and to utilize N-methyl-D-aspartate (NMDA) receptors. These assumptions were tested here in opioid receptor triple knock-out (KO) mice lacking all three genes encoding opioid receptors (mu, delta, and kappa) by comparing their thermal nociceptive responses to the opioids morphine and oxymorphone with those of B6129F(1) controls. Injecting acute opioid bolus doses in controls caused maximal analgesia that was completely abolished in KO mice, confirming the functional consequence of the KO mouse opioid receptor deficiency. Continuous opioid infusion by osmotic pump in control mice also initially caused several consecutive days of analgesia that was shortly thereafter followed by several consecutive days of hyperalgesia. In contrast, continuously infusing KO mice with opioids caused no detectable analgesic response, but only immediate and steady declines in nociceptive thresholds culminating in several days of unremitting hyperalgesia. Finally, injecting the non-competitive NMDA receptor antagonist MK-801 during opioid infusion markedly reversed hyperalgesia in control but not KO mice. These data demonstrate that sustained morphine and oxymorphone delivery causes hyperalgesia independently of prior or concurrent opioid or NMDA receptor activity or opioid analgesia, indicating the contribution of mechanisms outside of current conceptions, and are inconsistent with proposals of hyperalgesia as a causative factor of opioid analgesic tolerance.

Assessment of acute and chronic morphine dependence in male and female mice

The present study compared male and female mice for frequency of naloxone-precipitated jumping and naloxone ED(50) values, two common indices of physical dependence, following acute and chronic morphine administration. Both sexes displayed a positive dose-response relationship between acute morphine and naloxone doses and jumping frequency. There was a significant main effect of sex, with mean jumping frequencies greater in males. The naloxone ED(50) estimate was also fourfold lower in males, indicating greater withdrawal sensitivity than females. Jumping frequencies were similar in male and female saline-treated control mice, discounting initial sex differences as a significant factor in the unequal magnitude and sensitivity in acute morphine dependence between sexes. In contrast, males and females displayed similar mean withdrawal jumping frequencies and naloxone ED(50) values after 3 days of morphine injections. Sex difference in withdrawal jumping was also not observed when morphine treatment was increased to 7 days via daily injection or continuous subcutaneous infusion. The present study demonstrates the development of greater physical dependence in male relative to female mice following acute but not chronic morphine administration.

Sex-specific mediation of opioid-induced hyperalgesia by the melanocortin-1 receptor.

Background:N-Methyl-d-aspartate receptor antagonists reverse hyperalgesia during morphine infusion in male mice only. Because the melanocortin-1 receptor can act as a female-specific counterpart to N-methyl-d-aspartate receptors in &kgr;-opioid analgesic mechanisms, the authors assessed the contribution of melanocortin-1 receptors to the sex-specific mechanisms underlying morphine hyperalgesia. Methods:The tail-withdrawal test was used to compare the nociceptive responses of male and female C57BL/6J (B6) mice with those of C57BL/6J-Mc1re/e (e/e) mice, spontaneous mutants of the B6 background lacking functional melanocortin-1 receptors, during continuous morphine infusion (1.6 and 40.0 mgkg−1 · 24 h−1). Separate groups of hyperalgesic B6 and outbred CD-1 mice were injected with MK-801 or MSG606, selective N-methyl-d-aspartate and melanocortin-1 receptor antagonists, respectively. Results:Morphine infusion (40.0 mg · kg−1 · 24 h−1) reduced baseline withdrawal latencies by 45–55% in B6 mice of both sexes, indicating hyperalgesia; this increased nociception was manifest in male e/e mice only. Although MK-801 reversed hyperalgesia in male mice only, increasing latencies by 72%, MSG606 increased latencies by approximately 60% exclusively in females. A lower morphine infusion dose (1.6 mg · kg−1 · 24 h−1) reduced baseline withdrawal latencies by 45–52% in B6 and e/e mice of both sexes, which was reversed by MK-801, but not MSG606, in both male and female B6 mice. Conclusions:The data indicate the sex-specific mediation of high-dose morphine-induced hyperalgesia by N-methyl-d-aspartate and melanocortin-1 receptors in male and female mice, respectively, suggesting a broader relevance of this known sexual dimorphism. The data further indicate that the neural substrates contributing to hyperalgesia are morphine dose-dependent.

Morphine hyperalgesia in mice is unrelated to opioid activity, analgesia, or tolerance: evidence for multiple diverse hyperalgesic systems.

Hyperalgesia following chronic morphine treatment is thought to be a response to opioid receptor activation and analgesia and contribute to the development of analgesic tolerance. Here, the relationship between these variables was studied in mice tested for nociceptive sensitivity on the tail-withdrawal test during chronic infusion of various morphine doses. Hyperalgesic onset was preceded by dose-dependent analgesia except for the lowest morphine dose, which caused hyperalgesia 6 h after the start of infusion. Morphine ED50 values obtained at various infusion intervals demonstrated both analgesic tolerance in the absence of hyperalgesia and hyperalgesia in the absence of tolerance. Continuous opioid receptor antagonism using naltrexone pellets abolished analgesia during continuous morphine administration, transiently potentiated hyperalgesia, and revealed differences in hyperalgesic onset between morphine infusion doses. Acute injection of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 attenuated hyperalgesia in naltrexone-treated mice, demonstrating a role for this receptor in morphine hyperalgesia unrelated to its effects upon morphine analgesia. In mice where hyperalgesia subsided after continuous infusion of the highest morphine dose (i.e., hyperalgesic adaptation), hyperalgesia was restored after infusing the lower but not higher morphine dose. In addition, acute injection of morphine-3beta-glucoronide (M3G) caused hyperalgesia that was cross-adaptive with the lower morphine dose only. The data demonstrate that morphine hyperalgesia is independent of prior or concurrent opioid receptor activity or analgesia and is unrelated to analgesic tolerance. Furthermore, the lack of hyperalgesic cross-adaptation between high and low morphine doses, and their differential cross-adaptation with M3G hyperalgesia, also suggests distinct morphine dose-dependent hyperalgesic systems.

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.

Mapping of a quantitative trait locus for morphine withdrawal severity

Chronic morphine exposure results in physical dependence, manifested by physical symptoms during naloxone-precipitated withdrawal. Jumping frequency is widely considered the most sensitive and reliable index of withdrawal intensity in mice. Inbred mouse strains surveyed for naloxone-precipitated withdrawal display large and significant strain differences in jumping frequency, including an approximately tenfold difference between C57BL/6 and 129P3 mice. In the present study, (B6 × 129)F2 hybrid mice were given daily morphine injections for four days using an escalating dosing schedule, and naloxone-precipitated withdrawal on day 5 was measured. A full-genome scan for linkage to phenotypic data was performed using polymorphic microsatellite markers. Significant linkage was observed between withdrawal jumping frequencies and a 28 cM-wide region of Chromosome 1 (32–60 cM; peak at 51 cM), accounting for 20% of the overall phenotypic variance. Two other suggestive QTLs were found, on Chromosomes 5 and 10, and an additive model fitting all three loci accounted for 43% of the total variance. F2 mice were also assessed for changes in morphine analgesic potency using the tail-withdrawal test in dose–response studies on days 1 and 4. No linkage was observed between Chromosomes 1, 5, and 10 and morphine analgesic tolerance, suggestive of genetic dissociation of naloxone-precipitated withdrawal from morphine and chronic morphine intake per se. The significant quantitative trait locus for naloxone-precipitated withdrawal severity in morphine-dependent mice, which we name Depmq1, may prove to be of considerable heuristic value once the underlying gene or genes are identified.

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.

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.