Research reportQuantitative autoradiographic mapping of μ-, δ- and κ-opioid receptors in knockout mice lacking the μ-opioid receptor gene
Introduction
Opioid peptides and their receptors are abundant in the central nervous system and play a major neuromodulatory role in the control of nociceptive stimuli and several other behavioural responses 6, 31. There is substantial evidence to suggest the existence of multiple opioid receptor types in the brain and three major classes (μ-, δ- and κ-) have been identified [10]. Each receptor type displays a unique distribution pattern in both the rat 21, 25, 40, 44and the mouse 30, 37and activation of each receptor produces distinct pharmacological responses [27]. The anatomical distribution of both the μ- and δ-receptors in areas controlling olfaction, visual, auditory and nociceptive processing implies a role in sensory integration, whilst the predominance of κ-receptors in the hypothalamus and median eminence point to a major role in neuroendocrine regulation [21]. Further, all three receptors are abundant in limbic regions where they mediate the mood altering properties of opioids 5, 20.
There is evidence for further subdivision of all three primary receptor types including μ1- and μ2- 32, 33δ1- and δ2- 4, 16, 24, 38and κ1-, κ2- and κ3- 2, 3, 42, 45. Also, there is some indication of co-operativity between each class most notably for the μ- and δ-opioid receptors 26, 36, 43.
Three genes have been cloned [17]and genetic approaches are now available to delineate the contribution of each receptor to opioid function in vivo. We have recently disrupted, by homologous recombination, the mouse μ-opioid receptor (MOR) gene [23]. Animals deficient in this receptor show no overt behavioural abnormalities or major compensatory changes in opioid peptide expression, but exhibit complete loss of analgesic and dependence responses to morphine [23]. Further, it appeared that no behavioural responses to morphine mediated by δ- or κ-opioid receptors could be detected since in some tests morphine should have activated these receptors also. Other groups have confirmed the lack of analgesic [39]and locomotor [41]responses to morphine in μ-knockout mice, although doses above 50 mg/kg were able to illicit a very small analgesic response in tail-flick tests [39]. In addition these studies [39]showed changes in nociceptive latencies in spinal analgesic tests in contrast to our studies [23]which found no change. Although whole-brain homogenate binding studies indicated no change in the maximal binding capacities for δ- and κ-receptors 23, 39the behavioural data raise questions over the possibility that the absence of the μ-receptor may produce subtle changes in the expression of other opioid receptor types and alter their anatomical distribution. We have therefore carried out a complete quantitative autoradiographic mapping, using highly selective radioligands, on coronal and sagittal sections to assess opioid receptor changes in wild-type, heterozygous and homozygous mice deficient in the μ-receptor.
Section snippets
Generation of knockout mice
The strategy for the development of mice deficient in the μ-opioid receptor gene has been described in detail elsewhere [23]. All mice studied were between 6 and 8 weeks of age and of mixed sexes. In all groups there were no indications of any quantitative differences between males and females and data from both sexes was pooled.
Autoradiographic procedures
Mice were killed by decapitation and intact brains removed and immediately frozen in isopentane at −20°C. Brains were stored at −70°C for a maximum period of 1 month
Results
Coronal mapping of μ-, δ-, κ1- and total κ-receptors is shown in Fig. 1 (with major structures labelled) and shows a distinct anatomical distribution of μ-, δ-, and κ-opioid receptors in mouse brain. There were no qualitative differences in the anatomical distribution of κ1- and total κ-receptors. This distribution pattern was also confirmed from sagittal autoradiography (Fig. 4, left column).
Discussion
[3H]DAMGO, [3H]DELT I and [3H]CI-977 were chosen to selectively label μ-, δ- and κ1-receptors not only because of their receptor selectivity (500–3000-fold, 7, 8, 15, 18, 46) but also because all three ligands have extremely low non-specific binding and, at the development times used in this study, non-specific binding images were undetectable for μ- and δ-binding and extremely faint for κ1-binding. It is clear from the complete lack of μ-receptors throughout all brain regions that the knockout
Conclusion
Quantitative autoradiographic mapping of opioid receptor subtypes has been carried out in the brains of wild-type, heterozygous and homozygous mutant mice deficient in the MOR gene to demonstrate the deletion of the MOR gene, to investigate the possible existence of any μ-receptor subtypes derived from a different gene and to determine any modification in the expression of other opioid receptors. μ-, δ-, κ1- and total κ-receptors, in adjacent coronal sections in fore- and midbrain and in
Acknowledgements
This work was funded by the Centre National de la Recherche Scientifique and Association pour la Recherche sur le Cancer. S.J. Slowe received a Pfizer-funded Studentship. B.K. received support from the Ministère de la Recherche et de la Technologie and the Institut Universitaire de France; I.K. and B.K. received support from the Biomed 2 programme of the European Community.
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