Unique behavioural phenotypes of recombinant-inbred CXBK mice: partial deficiency of sensitivity to μ- and κ-agonists
Introduction
CXBK mouse strain is a recombinant-inbred strain derived by full-sib mating from a cross between C57BL/6By and BALB/cBy mice (Bailey, 1971). Since the discovery by Baran et al. (1975) that CXBK mice had a low level of opioid receptors in the brain and showed insensitivity to morphine, the mouse strain has been used as a μ-opioid-receptor deficient strain in a variety of investigations. The important role of opioids in electroacupuncture analgesia and stress-induced analgesia has been suggested by lower levels of electroacupuncture analgesia (Peets and Pomeranz, 1978) and lower levels of analgesia following defeat or swimming in CXBK mice (Miczek et al., 1982, Marek et al., 1988). Following systemic or intracerebroventricular (i.c.v.) administration, the morphine ED50 for inducing analgesia in the tail-flick test in CXBK mice was greater than 10 times that in C57BL/6 mice (Moskowitz and Goodman, 1985, Vaught et al., 1988). In contrast, i.c.v. administration of a δ agonist was equipotent in both CXBK and C57BL/6 mice in inducing analgesia in the tail-flick test (Vaught et al., 1988), and no reduction of the analgesic effects of κ agonists was shown using the abdominal constriction assay, following systemic administration (Muraki et al., 1991, Quock et al., 1993). Thus, the genetic alteration in CXBK mice seems to exist in the μ-opioid-receptor gene, although no molecular biological evidence has ever been reported.
On the other hand, μ-opioid-receptor-deficient (μKO) mice have been recently produced by gene-targeting (Matthes et al., 1996, Sora et al., 1997b, Tian et al., 1997, Loh et al., 1998). The μKO mice showed neither the analgesic effect of morphine, preference for morphine-associated places, nor physical dependence on morphine (Matthes et al., 1996). The morphine ED50 for inducing analgesia in the tail-flick and hot-plate tests in μKO mice was reported to be more than 400 mg kg−1 (Loh et al., 1998). Not only morphine but also DPDPE, a selective δ-opioid-receptor agonist, failed to induce analgesia in μKO mice in spite of normal expression of the δ-opioid receptor in the mice (Sora et al., 1997a). As expected from previous reports of a low nociceptive threshold in naloxone-treated mice (Roques et al., 1993, for review), the nociceptive threshold in μKO mice was lower (Sora et al., 1997b). Neither analgesia nor hypolocomotion was induced by U-50488, a κ-opioid-receptor agonist, in κ-opioid-receptor-deficient (κKO) mice produced by Kieffer and her colleagues (Simonin et al., 1998). However, morphine-induced analgesia in κKO mice was equivalent to that observed in other mice.
Although the behavioural phenotypes of CXBK mice have been extensively investigated, it remains to be elucidated whether or not the phenotypes of CXBK mice are similar to either of the μKO or κKO mouse phenotypes. In this study, we investigated the response to heat stimuli and spontaneous locomotion in opioid-treated and untreated CXBK mice with the same tests as employed for the gene-targeting mice, in the hope of understanding the similarities and differences in behavioural phenotypes between CXBK mice and the gene-targeting mice. We found that the CXBK mouse phenotypes are different from both the μKO and κKO mouse phenotypes. Furthermore, we obtained unexpected findings of the analgesic and sedative effects of U-50488 being reduced and the nociceptive threshold being lowered in CXBK mice. Based on the present results, we discuss the gene responsible for the CXBK mouse phenotype, the cross-talk of signals mediated by μ- and κ-opioid receptors in vivo, and the μ-opioid-receptor subtypes.
Section snippets
Animals
Adult (>6 weeks old) mice were used in all the experiments. Each naive mouse was only used once. Subjects were housed with littermates of the same sex (up to five mice per cage) in aluminium cages, and maintained on a 12-h, 12-h dark cycle (lights on 07:00–19:00 h) at 23±1°C under a relative humidity of 50±5%. The mice had free access to a standard commercial laboratory diet (NMF, Oriental Yeast Co., Ltd., Tokyo, Japan) and water. CXBK mice were originally purchased from Jackson Laboratory (ME,
Partial deficiency of sensitivity to morphine in CXBK mice
The analgesic effects of morphine on C57 and CXBK mice were compared using the tail-flick test (Fig. 1a) and hot-plate test (Fig. 1b). In the tail-flick test, CXBK mice administered 3 and 10 mg kg−1 morphine (i.p.) responded to the heat stimulus with short latencies as saline-administered CXBK mice did, whereas C57 mice administered 3 mg kg−1 morphine responded with longer latencies than saline-administered C57 mice, and none of C57 mice administered 10 mg kg−1 morphine responded by the 15-s
Difference in phenotype between CXBK mice and the μKO and κKO mice
Although CXBK mice have been used as μ-opioid-receptor deficient mice for a variety of investigations, there has been no molecular biological evidence of a gene deficiency, and CXBK mice have never been compared with gene-targeting mice lacking the opioid receptor. The detailed behavioural phenotyping of CXBK mice in this study made it possible to compare the CXBK and gene-targeting mouse phenotypes. The apparent morphine analgesia at doses of 30 and 100 mg kg−1 in CXBK mice (Fig. 1) was in
Conclusion
We found in this study that the sensitivity to morphine and U-50488 was lowered in CXBK mice, and that the threshold for heat nociception was also lowered in these mice. The findings provide behavioural evidence that CXBK mice exhibit different phenotypes from C57, μKO and κKO mouse phenotypes. Identification of the specific genetic alterations underlying the unique CXBK phenotype may lead to further understanding of the opioid system, such as the cross-talk of signals mediated by opioid
Acknowledgements
We wish to thank Dr Hiroaki Niki (Laboratory for Neurobiology of Emotion, RIKEN) for critical reading and discussion, Mr Tsutomu Oowada (Laboratory for Animal Research Center, RIKEN) for animal breeding and care, and Ms Naomi Yamazaki, Ms Tazuko Kiyosaki, and Ms Chikako Honma in our laboratories for assistance. This investigation was supported by research grants from Cooperative Research Program of the RIKEN Brain Science Institute and Special Postdoctoral Researchers Program, RIKEN, and the
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