In the G‐protein‐coupled receptor superfamily, the opioid receptor subfamily is constituted of the three distinct opioid receptors (namely δ‐, μ‐ and κ‐subtypes) and the receptor for nociceptin (also designated orphaninFQ). The members of the opioid receptor subfamily were known to mediate a variety of cellular inhibitory effects. The three opioid receptors are known to play central roles in mediating analgesia and many other physiological activities; however, the nociceptin receptor was identified recently and less is known about its physiological roles. Here we report the generation and characterization of mice lacking the nociceptin receptor. The knockout mice showed no significant differences in nociceptive threshold and locomotor activity compared with control mice, but they lost nociceptin‐induced behavioral responses. These results indicate that the nociceptin system is not essential for regulation of nociception or locomotor activity. On the other hand, we found insufficient recovery of hearing ability from the adaptation to sound exposure in the mutant mice. Thus, the nociceptin system appears to participate in the regulation of the auditory system.
Multiple binding sites for opioid ligands have been identified based on pharmacological studies, and the opioid receptors have been shown to mediate a variety of cellular inhibitory effects, including inhibition of adenylate cyclase, activation of potassium channels and inhibition of calcium channels (Loh and Smith, 1990). Previous DNA cloning studies have shown that in the G‐protein‐linked receptor superfamily (Hille, 1992) the opioid receptor subfamily is constituted of three opioid receptors (designated as δ‐, μ‐ and κ‐subtypes) and an opioid receptor homolog, termed ORL1, ROR‐C or LC132 (Bunzow et al, 1994; Fukuda et al., 1994; Mollereau et al., 1994; Knapp et al., 1995). Despite a high degree of amino acid sequence similarity between the members of the opioid receptor subfamily, the opioid receptor homologs expressed from the cDNAs did not show high affinities for opioid ligands. Recently, nociceptin, also designated orphaninFQ, has been identified as a novel peptide agonist for the opioid receptor homolog from the brain (Meunier et al., 1995; Reinscheid et al., 1995). Nociceptin is derived from a larger precursor which shows sequence similarity to the opioid peptide precursors, particularly pre‐prodynorphin (Houtani et al., 1996; Mollereau et al., 1996; Nothacker et al., 1996). Thus, the nociceptin system is closely related to the opioid peptide system structurally, evolutionarily and functionally.
So far, several groups have reported the biological functions of the nociceptin system. In contrast to the opioid peptides with analgesic effects, nociceptin induced hyperalgesia or allodynia, when administered intracerebroventricularly (i.c.v.) or intrathecally, respectively (Meunier et al., 1995; Okuda‐Ashitaka et al., 1996). Nociceptin caused a decrease in locomotor activity (Reinscheid et al., 1995). Inhibition of expression of the nociceptin receptor reduced immunoglobulin production, and a mitogen induced expression of nociceptin receptor mRNA in lymphocytes (Halford et al., 1995; Wick et al., 1995). Expression of nociceptin precursor mRNA was regulated by intracellular cAMP levels in NS20Y neuroblastoma cells (Saito et al., 1996). The reported results suggested important roles for the nociceptin system in modulation of the nociceptive threshold, locomotor activity, immunological responses and neuronal development. However, specific antagonists for the nociceptin receptor are not available, and the physiological roles of the nociceptin system have not been elucidated yet at the whole‐animal level. One approach to this issue would be to produce mutant mice lacking the nociceptin receptor by means of the gene targeting technique and to analyze the physiological phenotypes of the mutants. We report here the generation of mice with a targeted mutation in the nociceptin receptor gene. Although the mutant mice showed no abnormalities in nociception, locomotion and the immune system, we found abnormal hearing ability in the mutants. The results clearly demonstrated an essential role for the nociceptin system in regulation of the auditory system.
Results and discussion
Generation of mutant mice lacking the nociceptin receptor
As shown in Figure 1A, genomic DNA fragments from the mouse nociceptin receptor gene (Nishi et al., 1994) were used to construct a targeting vector, in which the first protein‐coding sequence (P1) is replaced with a lacZ–neo cassette and the sequence of the lacZ gene encoding bacterial β‐galactosidase is linked to the 5′‐non‐coding sequence. In the expected mutant allele, it is thus possible to express the lacZ gene under the control of the transcription machinery of the nociceptin receptor gene. The gene targeting and generation of mutant mice were performed essentially as described previously (Takeshima et al., 1994); in the experimental procedures, we used J1 embryonic stem (ES) cells derived from a 129 strain mouse (Li et al., 1992) and C57BL/6J mice to cross with chimeric males. Two lines of mice carrying the mutant allele designated as morcm1 were established using independent ES clones positive in Southern blot analysis (Figure 1B).
RNA blot hybridization analysis failed to detect mRNA for the nociceptin receptor in the brains of the homozygous mice (Figure 2A); therefore, the morcm1 mutation seems to be a null mutation in the gene. On the other hand, the expression levels of nociceptin precursor mRNA are comparable in the brains of mutant and control mice (Figure 2B). The homozygous mice obtained were healthy and fertile, and we could not find any anatomical abnormalities in the mutants. Two lines of the nociceptin receptor‐deficient mice yielded essentially the same results in the experiments described below.
Nociception in mice lacking the nociceptin receptor
Because hyperalgesia and allodynia induced by nociceptin have been reported (Meunier et al., 1995; Okuda‐Ashitaka et al., 1996), we conducted the heat tail‐flick test to compare nociceptive thresholds in the nociceptin receptor‐deficient and control mice (Figure 3A and B). If the nociceptin system functioned as a major regulator in the nociceptive system, the loss of the nociceptin receptor might result in anti‐nociceptive effects in the mutant mice. In accordance with the reported data, nociceptin induced hyperalgesia when administered i.c.v. to wild‐type mice. In contrast, we could not find a significant difference in the latency of the tail‐flick response to heat between the untreated homozygous mutants and those treated with nociceptin. However, no measurable difference in nociceptive thresholds was observed between the untreated mutant and control mice under different conditions of heat intensity (Figure 3A and B). Moreover, we also examined nociceptive thresholds in the test using acetic acid‐induced writhing behavior, a test that allows a more sensitive detection of the thresholds than the tail‐flick test (Figure 3C). Again the mutant mice showed nociceptive thresholds comparable with that of control mice.
The results clearly show that nociceptin‐induced hyperalgesia is mediated by the product derived from the nociceptin receptor gene, and also indicate that regulation by the nociceptin system is not essential for the determination of the nociceptive threshold in vivo. However, our data cannot eliminate the possibility that neuromodulation systems other than the nociceptin system, for example opioid peptide systems, may compensate for abnormalities in the mutant mice. On the other hand, the loss of the nociceptin system does not affect the morphine‐induced anti‐nociceptive response (Figure 3B). Recently, it was reported that mutant mice lacking the μ‐opioid receptor lost morphine‐induced anti‐nociceptive effects but showed an apparently normal nociceptive threshold (Matthes et al., 1996). Together these observations may suggest that the nociceptive threshold is regulated by highly compensating mechanisms in the central nervous system.
Locomotion in mice lacking the nociceptin receptor
The spontaneous locomotor activity of the mutant mice was then examined because reduction of locomotor activity by nociceptin administration was observed (Reinscheid et al., 1995). Assuming that the nociceptin system was a dominant modulation system in locomotor activity, hyperactivities might be expected in the mutant mice. However, the results showed a slightly reduced locomotor activity of the homozygous mutants as compared with that of control mice (Figure 4). We also examined the effects of nociceptin administration on locomotor activity. Nociceptin‐induced hypoactivity was observed in wild‐type mice as reported previously, but we could not detect any obvious effect of nociceptin in the homozygous mutants (Figure 4). These results show that disruption of the nociceptin receptor gene results in the loss of nociceptin‐induced hypolocomotion.
The results of nociceptin‐induced hypolocomotion in wild‐type mice and the tendency to hypolocomotion in the homozygous mutants seem to be mutually exclusive. However, these results could be compatible because locomotor activity is controlled by highly complex neural circuits interconnecting the sensory and motor systems. Alternatively, it may be that the reduced locomotor activity in the homozygous mutants is due to the genetic background rather than the loss of the nociceptin receptor. In our targeting procedures, the genetic background of the 129 strain rather than the C57BL mouse would have the predominating influence in the homozygous mutant. Strain 129 mice exhibit a significant locomotor hypoactivity in comparison with C57BL mice, and the majority of knockout mice with the mixed 129–C57BL genotype exhibit hypoactivity (Gerlai, 1996). Nevertheless, the absence of any obvious abnormality in locomotor activity of the mutant mice suggests that the nociceptin system is not a major determinant in the regulation of locomotion in the central nervous system.
The immune system in mice lacking the nociceptin receptor
It was reported that antisense oligonucleotides for the nociceptin receptor inhibit immunoglobulin production in B‐lymphocytes and that mitogen stimulation resulted in a remarkable induction of the level of nociceptin receptor mRNA in peripheral blood lymphocytes (Halford et al., 1995; Wick et al., 1995). These observations may suggest important immunological roles for the nociceptin system. However, we detected no abnormalities in the immunoglobulin content of peripheral blood from nociceptin receptor‐deficient mice (data not shown). Moreover, in flow cytometric analysis using antibodies to specific cell surface markers, we identified no abnormal populations of T‐ and B‐lymphocytes in the blood and bone marrow (data not shown). These results suggest that the nociceptin system is not essential for lymphocyte proliferation and immunoglobulin production.
Regional distribution of the nociceptin receptor and nociceptin precursor
As expected from the targeting construct, neurons expressing the nociceptin receptor can be detected by β‐galactosidase activity in the mutant mice. Indeed, the regions positive in X‐gal staining in brain and spinal cord from mutants (Figure 5A–G) almost correspond with the areas positive in previous in situ experiments with antisense probes for the nociceptin receptor mRNA (Fukuda et al., 1994; Mollereau et al., 1994). Furthermore, the brain and spinal cord areas with predominant expression of the nociceptin precursor message (Figure 5H–K) appeared identical to the sites that exhibit significant levels of β‐galactosidase activity (Figure 5E–G). These results, together with our previous observations (Houtani et al., 1996), indicate that the nociceptin receptor in these sites may be located on, or afforded many synapses by, nociceptin neurons. They also imply that the nociceptin system may modulate a variety of neurons in central nervous system sites, including the sensory and auditory pathways and centers implicated in emotion and autonomic control.
Hearing ability in mice lacking the nociceptin receptor
With the evidence for nociceptin and its receptor in brainstem auditory stations as shown above, we then examined the hearing ability of the mutant mice. Morphologically, the middle and inner ears and acoustic nerve of the mutant mice appeared normal (data not shown). The auditory brainstem responses (ABRs) measured in untreated mutant mice showed normal waveforms and thresholds (average threshold value: 37 dB SPL), which are comparable with those obtained in control mice (Colvin et al., 1996). However, following exposure to intense sound, the thresholds of ABRs in the homozygous mutants showed a significant rise (Figure 6A). The threshold shift was considered temporary because the ABRs recovered completely to normal levels within 3 days (Figure 6B). Neither C57BL nor 129 strain mice showed the threshold shift. It is unlikely, therefore, that the shift is due to the heterogeneity in the genetic background. The results indicate that the nociceptin system is essential for the regulation of hearing ability following exposure to sound.
The mechanism of the disregulation remains to be investigated. We have shown abundant nociceptin precursor messages in the periolivary region (Figure 5), which is the principal site of the origin of efferent fibers, termed the crossed olivocochlear bundle, to the cochlear hair cells (Vetter et al., 1991; Brown, 1993; Simmons et al., 1996). Unlike other sensory systems, these efferent neurons present outstanding features for the auditory system because they have pivotal roles in directly influencing both the central auditory nuclei and the periphery (Brown, 1993). Stimulation or surgical impairment of the olivocochlear bundle has been shown to produce changes in cochlear mechanics, afferent fiber activities and efficiencies of signal detection (Dewson, 1968; Borg, 1971; Klinke and Galley, 1974; Mountain, 1980; Cody and Johnstone, 1982). The nociceptin system may modulate the excitability of the olivocochlear bundle and/or cochlear hair cells for the regulation of the auditory system. The present results provide a model system suitable for the investigation of the molecular mechanism of hearing ability and sound injury.
Materials and methods
Generation of mutant mice
The targeting vector was constructed using the genomic DNA fragments derived from λMORG5 (Nishi et al., 1994), a synthetic polylinker carrying SacI, XbaI and HindIII sites, the 3.7 kb HindIII–BamHI fragment from pCH110 (Pharmacia Biotec.), the 1.1 kb XhoI–BamHI fragment from pMC1 Neo poly(A) (Stratagene) and the ∼3 kb XhoI–SalI fragment containing the virus thymidine kinase gene (Takeshima et al., 1994) and pBluescript SK(−) (Stratagene). The short arm of the vector is the 2.0 kb SpeI–RsaI fragment containing the putative promoter and 5′‐untranslated sequences, and the long arm is the 8.5 kb SpeI–SalI (vector) fragment containing the second and third protein‐coding sequences. The vector was linearized with NotI and transfected into J1 ES cells (Li et al., 1992). Of ∼250 clones screened by Southern blotting, we identified four clones carrying the homologous mutation. Chimeric mice produced with two clones (numbered 301 and 378) of the positive ES clones could transmit the mutation to their pups. Young adult mice (9–12 weeks old) were used for the analyses in this report. Blot hybridization analyses of DNA and RNA preparations from tissues of adult mice were performed as described previously (Takeshima et al., 1994).
Male mice were used for behavioral analyses. Analgesia was determined using the radiant heat, tail‐flick technique (D'Amour and Smith, 1941). The latency to withdraw the tail from a focused light stimulus was measured electronically, using a photocell. For analyzing the effects of nociceptin, the latencies were measured 10 min after administration of nociceptin (1 or 10 nmol, i.c.v.) and were compared with baseline latencies. To examine the effects of morphine (10 mg/kg, s.c.) the latencies in mice were determined 60 min after administration. We used a higher intensity of light stimulation to examine the morphine‐induced anti‐nociceptive effects and a lower intensity to analyze the nociceptin‐induced hyperalgesic effects. The maximum latency to response of 15 and 20 s would not induce tissue damage in mice treated with morphine and nociceptin, respectively.
Mice tested in the writhing assay received an injection (10 ml/kg, i.p.) of 0.7% acetic acid. The number of writhes, characterized by a wave of contraction of the abdominal muscles followed by extension of the hind limbs, was counted for 10 min, beginning 5 min after the injection.
To determine locomotor activity (Noda et al., 1995), each animal was placed individually in a transparent acrylic cage (26×44×40 cm) and the locomotor count was recorded over 30 min using digital counters with infrared cell sensors placed on the walls (SCANET SV‐10, Toyo‐sangyo Co., Japan). To examine the effects of nociceptin, mice were injected with nociceptin (1 or 10 nmol, i.c.v.) and 10 min later the locomotor activities of the mice were measured. Experiments were carried out from 11:00 to 15:00.
For β‐galactosidase staining, mice were perfused with a 2% paraformaldehyde solution buffered with 0.12 M sodium phosphate (pH 7.4) under pentobarbital anesthesia, and the brains and spinal cords were immersed immediately in ice‐cold 25% sucrose. Frozen coronal sections (40 μm thickness) were stained with the X‐Gal reagent (Bajocchi et al., 1993). For in situ hybridization, mice were perfused with a fixative containing 4% paraformaldehyde, 2% glutaraldehyde and 0.12 M sodium phosphate (pH 7.4). Brains and spinal cords were cut into coronal sections (50 μm thickness) and analyzed with the probe for nociceptin precursor mRNA as described previously (Houtani et al., 1996).
Auditory brainstem responses
ABRs were measured (Colvin et al., 1996) before and after sound exposure using chloral hydrate‐administered mice by means of stepwise 0.1 ms click sound stimulations. Intense sound (1 kHz continuous pure tone at 110 dB SPL for 60 min) was applied to the mice in a soundproof room.
We thank Misa Shimuta, Ichiro Saito, Yoshinobu Sugitani and Hitomi Yamanaka for help in some experiments. This work was supported in part by Grants from the Ministry of Education, Science, Sports and Culture, the Japan Private School Promotion Foundation, the Naito Foundation, the Uehara Memorial Foundation, the Japanese Foundation of Metabolism and Diseases, the Ichiro Kanehara Foundation and the Mitsubishi Foundation.
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