Brigitte L. Kieffer

Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses.

The role of the opioid system in controlling pain, reward and addiction is well established, but its role in regulating other emotional responses is poorly documented in pharmacology. The μ-, δ- and κ- opioid receptors (encoded by Oprm, Oprd1 and Oprk1, respectively) mediate the biological activity of opioids. We have generated Oprd1-deficient mice and compared the behavioural responses of mice lacking Oprd1, Oprm (ref. 6) and Oprk1 (ref. 7) in several models of anxiety and depression. Our data show no detectable phenotype in Oprk1−/− mutants, suggesting that κ-receptors do not have a role in this aspect of opioid function; opposing phenotypes in Oprm−/− and Oprd1−/− mutants which contrasts with the classical notion of similar activities of μ- and δ-receptors; and consistent anxiogenic- and depressive-like responses in Oprd1−/− mice, indicating that δ-receptor activity contributes to improvement of mood states. We conclude that the Oprd1-encoded receptor, which has been proposed to be a promising target for the clinical management of pain, should also be considered in the treatment of drug addiction and other mood-related disorders.

Opioids: first lessons from knockout mice

Opioid receptors of the mu-, delta- and kappa-subtypes mediate the potent analgesic and addictive actions of opioid drugs. They also regulate responses to pain, stress and emotions when activated by endogenous opioid peptides. Recently, mice lacking opioid receptors or opioid peptides have been produced by gene targeting, providing molecular tools to study opioid function in vivo. Observations on mutant mice have shed new light on the mode of action of opioids, opioid receptor heterogeneity and interactions, and the involvement of each component of the opioid system in mouse physiology. In this article, Brigitte L. Kieffer reviews the first reported studies and discusses their therapeutic implications.

Disruption of the κ‐opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective κ‐agonist U‐50,488H and attenuates morphine withdrawal

μ‐, δ‐ and κ‐opioid receptors are widely expressed in the central nervous system where they mediate the strong analgesic and mood‐altering actions of opioids, and modulate numerous endogenous functions. To investigate the contribution of the κ‐opioid receptor (KOR) to opioid function in vivo, we have generated KOR‐deficient mice by gene targeting. We show that absence of KOR does not modify expression of the other components of the opioid system, and behavioural tests indicate that spontaneous activity is not altered in mutant mice. The analysis of responses to various nociceptive stimuli suggests that the KOR gene product is implicated in the perception of visceral chemical pain. We further demonstrate that KOR is critical to mediate the hypolocomotor, analgesic and aversive actions of the prototypic κ‐agonist U‐50,488H. Finally, our results indicate that this receptor does not contribute to morphine analgesia and reward, but participates in the expression of morphine abstinence. Together, our data demonstrate that the KOR‐encoded receptor plays a modulatory role in specific aspects of opioid function.

Motivational Effects of Cannabinoids Are Mediated by μ-Opioid and κ-Opioid Receptors

Repeated THC administration produces motivational and somatic adaptive changes leading to dependence in rodents. To investigate the molecular basis for cannabinoid dependence and its possible relationship with the endogenous opioid system, we explored Δ9-tetrahydrocannabinol (THC) activity in mice lacking μ-, δ- or κ-opioid receptor genes. Acute THC-induced hypothermia, antinociception, and hypolocomotion remained unaffected in these mice, whereas THC tolerance and withdrawal were minimally modified in mutant animals. In contrast, profound phenotypic changes are observed in several place conditioning protocols that reveal both THC rewarding and aversive properties. Absence of μ receptors abolishes THC place preference. Deletion of κ receptors ablates THC place aversion and furthermore unmasks THC place preference. Thus, an opposing activity of μ- and κ-opioid receptors in modulating reward pathways forms the basis for the dual euphoric–dysphoric activity of THC.

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-, κ 1 - and total κ-receptors, in adjacent coronal sections in fore- and midbrain and in sagittal sections, were labelled with [ 3 H]DAMGO ( d -Ala 2 -MePhe 4 -Gly-ol 5 enkephalin), [ 3 H]DELTxa0I ( d -Ala 2 deltorphinxa0I), [ 3 H]CI-977 and [ 3 H]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-, κ 1 - 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.

Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus.

A random‐primed cDNA expression library constructed from the mRNA of neuroblastoma cells (NG108) was used to clone a specific rabies virus (RV) receptor. A soluble form of the RV glycoprotein (Gs) was utilized as a ligand to detect positive cells. We identified the murine low‐affinity nerve‐growth factor receptor, p75NTR. BSR cells stably expressing p75NTR were able to bind Gs and G‐expressing lepidopteran cells. The ability of the RV glycoprotein to bind p75NTR was dependent on the presence of a lysine and arginine in positions 330 and 333 respectively of antigenic site III, which is known to control virus penetration into motor and sensory neurons of adult mice. P75NTR‐expressing BSR cells were permissive for a non‐adapted fox RV isolate (street virus) and nerve growth factor (NGF) decreased this infection. In infected cells, p75NTR associates with the RV glycoprotein and could be precipitated with anti‐G monoclonal antibodies. Therefore, p75NTR is a receptor for street RV.

Distribution of nociceptin/orphanin FQ receptor transcript in human central nervous system and immune cells

We have examined the distribution of the opioid receptor-like-1 (ORL-1) transcript in the human CNS as well as human immune cells by RT-PCR and RNAse protection. The hORL-1 mRNA was distributed throughout the brain and particularly abundant in cortical areas, striatum, thalamus and hypothalamus. In the immune system, gene transcription was observed in normal circulating lymphocytes and monocytes as well as in T, B and monocytic cell lines. A splice variant, lacking 15 nucleotides at the junction between exon 1 and exon 2, showed a distribution similar to the already known ORL-1 transcript. Altogether these results show comparable expression levels of the hORL-1 gene in both nervous and immune systems, suggesting that the ORL-1-encoded receptor may participate to neuronal and non-neuronal physiological functions in humans.

Quantitative autoradiographic mapping of opioid receptors in the brain of δ-opioid receptor gene knockout mice

Using quantitative receptor autoradiography we have determined if deletion of the delta-opioid receptor gene (Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse delta-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. Delta-opioid receptors were labeled with [(3)H]deltorphin I (7 nM), mu- with [(3)H]DAMGO (4 nM), and kappa- with [(3)H]CI-977 (2.5 nM) or [(3)H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [(3)H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [(3)H]deltorphin I binding correlated significantly with those demonstrating high levels of the mu-receptor and were abolished in the presence of the mu-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [(3)H]DAMGO and [(3)H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [(3)H]bremazocine binding were higher in brains from -/- than +/+ mice. Our findings suggest that residual [(3)H]deltorphin I binding in the brain of delta-receptor gene knockout mice is the result of cross-reactivity with mu-sites and that there are no delta-receptor subtypes derived from a different gene. Changes in mu- and kappa-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the delta-receptor. The differences in [(3)H]CI-977 and [(3)H]bremazocine binding indicate these ligands show differential recognition of the kappa-receptor.

Lack of reward and locomotor stimulation induced by heroin in μ-opioid receptor-deficient mice

The micro-opioid receptor is the main substrate mediating opiate reward. Multiple micro-opioid receptor subtypes have been postulated to underlie opiate actions. Animals treated with antisense oligonucleotides targeting specific micro-opioid receptor exons show differential sensitivity to morphine versus heroin. The present work examined the rewarding and locomotor activating effects of heroin in mutant mice with a disrupted exon 2 of the micro-opioid receptor. Heroin (1-3 mg/kg) produced significant place preferences and stimulated locomotor activity in wild-type mice, whereas it had no effect in micro-opioid receptor-deficient mice. In contrast, treatment with cocaine (10-30 mg/kg) produced comparable place preferences and locomotor activation in both wild-type and micro-opioid receptor-deficient mice, thus providing evidence that the mutant mice are able to show drug-induced effects in the two behavioral paradigms used here. These results support an essential role for the micro-opioid receptor in the rewarding and locomotor activating effects of heroin.

Quantitative autoradiography of μ-,δ- and κ1 opioid receptors in κ-opioid receptor knockout mice

Abstract Mice deficient in the κ-opioid receptor (KOR) gene have recently been developed by the technique of homologous recombination and shown to lack behavioural responses to the selective κ1-receptor agonist U-50,488H. We have carried out quantitative autoradiography of μ-, δ- and κ1 receptors in the brains of wild-type (+/+), heterozygous (+/−) and homozygous (−/−) KOR knockout mice to determine if there is any compensatory expression of μ- and δ-receptor subtypes in mutant animals. Adjacent coronal sections were cut from the brains of +/+, +/− and −/− mice for the determination of binding of [ 3 H ]CI-977, [ 3 H ]DAMGO ( d -Ala2-MePhe4-Gly-ol5 enkephalin) or [ 3 H ]DELT-I ( d -Ala2 deltorphin I) to κ1-, μ- and δ-receptors, respectively. In +/− mice there was a decrease in [ 3 H ]CI-977 binding of approximately 50% whilst no κ1-receptors could be detected in any brain region of homozygous animals confirming the successful disruption of the KOR gene. There were no major changes in the number or distribution of μ- or δ-receptors in any brain region of mutant mice. There were, however some non-cortical regions where a small up-regulation of δ-receptors was observed in contrast to an opposing down-regulation of δ-receptors evident in μ-knockout brains. This effect was most notable in the nucleus accumbens and the vertical limb of the diagonal band, and suggests there may be functional interactions between μ- and δ-receptors and κ1- and δ-receptors in mouse brain.