Exploring the opioid system by gene knockout

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Abstract

The endogenous opioid system consists of three opioid peptide precursor genes encoding enkephalins (preproenkephalin, Penk), dynorphins (preprodynorphin, Pdyn) and β-endorphin (βend), proopiomelanocortin (POMC) and three receptor genes encoding mu-opiod receptor (MOR), delta-opiod receptor (DOR) and kappa-opiod receptor (KOR). In the past years, all six genes have been inactivated in mice by homologous recombination. The analysis of spontaneous behavior in mutant mice has demonstrated significant and distinct roles of each gene in modulating locomotion, pain perception and emotional behaviors. The observation of opposing phenotypes of MOR- and DOR-deficient mice in several behaviors highlights unexpected roles for DOR to be further explored genetically and using more specific delta compounds. The analysis of responses of mutant mice to exogenous opiates has definitely clarified the essential role of MOR in both morphine analgesia and addiction, and demonstrated that DOR and KOR remain promising targets for pain treatment. These studies also show that prototypic DOR agonists partially require MOR for their biological activity and provide some support for the postulated mu–delta interactions in vivo. Finally, data confirm and define a role for several genes of the opioid system in responses to other drugs of abuse, and the triple opioid receptor knockout mutant allows exploring non-classical opioid pharmacology. In summary, the study of null mutant mice has extended our previous knowledge of the opioid system by identifying the molecular players in opioid pharmacology and physiology. Future studies should involve parallel behavioral analysis of mice lacking receptors and peptides and will benefit from more sophisticated gene targeting approaches, including site-directed and anatomically-restricted mutations.

Introduction

Opium has been used for thousands of years because it produces euphoria and relieves pain. Morphine was isolated from the poppy seed and is used in the clinic for the treatment of severe pain, while heroin was synthesized by morphine diacetylation and has become one of the most addictive—and profitable—illegal drugs of abuse. It soon became clear that opiates modify brain functions in ways that can be useful when tightly controlled—or disastrous for the long-run user. Early efforts to understand the endogenous targets of opiate drugs provided receptor sites. Binding studies suggested three main classes of opioid receptors, currently named mu, delta and kappa receptors. Met- and Leu-enkephalin were then isolated and characterized as the first endogenous peptidic ligands for opioid receptors. Their discovery was soon followed by the identification of other peptides, including β-endorphin (βend) and dynorphins. Exogenous opiates therefore interact with an endogenous neurotransmitter system, which is critically implicated in the control of mood, motivation and pain.

The opioid system has been explored by the pharmacology for almost three decades. Morphine derivatives, and later agonists or antagonists with mu, delta or kappa selectivity, were used to show that the endogenous opioid system regulates (i) nociceptive information, with overlapping contributions of mu, delta and kappa receptors, (ii) hedonic homeostasis, mainly based on reinforcing properties of mu and delta agonists and aversive activity of kappa compounds, and (iii) stress responses with a main implication of mu and delta receptors. The pharmacological exploration also led to involve the opioid system in many other physiological responses, such as respiration, gastrointestinal motility, endocrine and immune functions. A tremendous amount of information has therefore accumulated (Vaccarino and Kastin, 2000), indicating that the opioid system is complex, and plays a central role in coping with threatening situations.

What are the components of the endogenous opioid system? Molecular approaches have identified opioid peptide precursor genes in the early 1980s, then genes encoding the receptors a decade later. Preproenkephalin (Penk) and preprodynorphin (Pdyn) genes encode several copies of enkephalins and dynorphins, respectively. The proopiomelanocortin (POMC) gene encodes βend, among other biologically active peptides. All these opioid peptides show the canonical Tyr-Gly-Gly-Phe-Met/Leu N-terminal sequence, indispensable to activate opioid receptors (Akil et al., 1997). More recently, distinct peptides named endomorphins have also been proposed as mu-selective endogenous opioids (Zadina et al., 1997). Three genes encoding the mu-opioid receptor (MOR), delta-opioid receptor (DOR) and kappa-opioid receptor (KOR) have been isolated and are otherwise referred as to Oprm, Oprd1 and Oprk1 using the mouse genome nomenclature, or MOP, DOP and KOP according to International Union of Pharmacology (IUPHAR). The three genes are highly homologous at the level of their predicted protein structure (Kieffer, 1995), and their genomic organization is almost identical (Gavériaux-Ruff and Kieffer, 1999), indicating that they probably evolved from a common ancestor. Opioid receptors belong to the G protein-coupled receptor family, and form a four-member gene subfamily together with the Orphanin FQ/nociceptin receptor discovered later (Darland et al., 1998).

Homologous recombination technology has opened the way to targeted gene mutagenesis in the early 1990s and mutant mouse strains lacking genes of the opioid system have been generated. These mice are extremely useful tools and their analysis nicely complements and extends pharmacological approaches. Opioid receptor-deficient mice allow now to accurately identify the molecular targets of prototypic opioid agonists or antagonists currently used by the pharmacologists. Most importantly, the precise phenotyping of each mutant strain leads to revisit the implication of each peptide and receptor in the many opioid-controlled behaviors and to discover functions that have remained unexplored. Initial data from MOR-, KOR-, DOR-, Penk- and βend-deficient mice have been reviewed earlier (Hayward and Low, 1999, Kieffer, 2000, Kieffer, 1999). At present, mice lacking every component of the opioid system have been generated, including Pdyn-deficient mice and many more aspects of opioid function have been explored in the knockout mice. Studies have been performed in several mutants for the same gene, or using mice mutated in distinct genes of the opioid system in similar studies. Opioid analgesia has been extensively investigated, and more sophisticated behaviors such as drug self-administration are being examined. Here we will review all the available information from the study of opioid peptide or receptor knockout mice, in an attempt to highlight what the genetic approach has added to our previous knowledge of the opioid system.

Finally, the opioid system interacts with many other neurotransmitter systems. Many null mutant mice for non-opioid genes have shown altered responses to morphine and these data have been overviewed elsewhere (Kieffer and Simonin, 2002).

Section snippets

Single opioid receptor knockout mice

Opioid receptor genes are organized similarly (Gavériaux-Ruff and Kieffer, 1999) and their coding regions extend over three exons encoding the extracellular domain and transmembrane domain I for exon 1, transmembrane domains II–IV for exon 2 and transmembrane domains V–VII followed by the cytoplasmic C-terminal region for exon 3. The MOR gene differs slightly in the 3′ coding region, where the last 12 codons are found on a fourth coding exon. Distinct targeting vectors have been constructed for

Behavioral phenotypes in the absence of drug

The successful generation of MOR, KOR and DOR, βend, Penk and Pdyn knockout mice, with mutations segregated at mendelian frequencies, indicates that the inactivation of genes of the opioid system is not lethal. Furthermore, the obtention of viable double MOR/DOR mutants or even triple MOR/DOR/KOR mutants (Gaveriaux-Ruff et al., 2001, Simonin et al., 2001) suggests that opioid receptors, either single or in combination, are not crucial for development, at least when animals are raised under home

Responses to opiates

During the several decades preceding molecular cloning of opioid receptors, enormous efforts have aimed at developing specific mu, delta and kappa agonists or antagonists to probe the opioid system. Pharmacological activity of this collection of “prototypic” opiates forms the basis for our current knowledge of opioid receptor function. Mice lacking opioid receptors represent ideal models to evaluate the molecular mechanism of action of prototypic opiates in a whole animal and under a large

Cannabinoids

Similarly to opioids, cannabinoids (CBs) induce antinociception, reward and dependence (Felder and Glass, 1998). In addition, pharmacological studies have provided strong evidence for interactions between CB and opioid systems (Manzanares et al., 1999). It was anticipated that mice lacking components of the opioid system would exhibit altered responses to CBs, just like mice lacking CB1 receptors showed altered responses to opioids (Ledent et al., 1999, Lichtman et al., 2001, Mascia et al., 1999

Concluding remarks and perspectives

Data using null mutant mice for genes encoding opioid peptides or receptors have now provided important findings on (i) the role of each component of the opioid system in mouse physiology and (ii) the respective contributions of MOR, DOR and KOR in responses to exogenous opiates in vivo. Phenotyping these animals in the absence of drugs has highlighted specific roles for receptors and peptides in various pain modalities, emotional behaviors and stress responses. The analysis of drug effects has

Acknowledgements

The authors wish to acknowledge the Human Frontier Science Program RG0011/2000-B, the Centre National de la Recherche Scientifique, The Université Louis Pasteur, The Association de la Recherche pour le Cancer, The Institut UPSA de la Douleur and the Mission Interministérielle de Lutte contre la Drogue et la Toxicomanie.

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