Quantitative autoradiographic mapping of μ-, δ- and κ-opioid receptors in knockout mice lacking the μ-opioid receptor gene

Abstract

Mice lacking the μ-opioid receptor (MOR) gene have been successfully developed by homologous recombination and these animals show complete loss of analgesic responses to morphine as well as loss of place-preference activity and physical dependence on this opioid. We report here quantitative autoradiographic mapping of opioid receptor subtypes in the brains of wild-type, heterozygous and homozygous mutant mice 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 sagittal sections, were labelled with [³H]DAMGO (d-Ala²-MePhe⁴-Gly-ol⁵ enkephalin), [³H]DELT I (d-Ala² deltorphin I), [³H]CI-977 and [³H]bremazocine (in the presence of DAMGO and DPDPE) respectively. In heterozygous mice, deficient in one copy of the MOR gene, μ-receptors were detectable throughout the brain at about 50% compared to wild-type. In brains from μ-knockout mice there were no detectable μ-receptors in any brain regions and no evidence for μ-receptors derived from another gene. δ-, κ₁- and total κ-receptor binding was present in all brain regions in mutant mice where binding was detected in wild-type animals. There were no major quantitative differences in κ- or δ-binding in mutant mice although there were some small regional decreases. The results indicate only subtle changes in δ- and κ-receptors throughout the brains of animals deficient in μ-receptors.

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.