Elsevier

Neuroscience

Volume 163, Issue 1, 29 September 2009, Pages 308-315
Neuroscience

Molecular Neuroscience
Research Paper
Complete knockout of the nociceptin/orphanin FQ receptor in the rat does not induce compensatory changes in μ, δ and κ opioid receptors

https://doi.org/10.1016/j.neuroscience.2009.06.021Get rights and content

Abstract

The nociceptin/orphanin FQ (N/OFQ) opioid peptide receptor (NOPr) is a new member of the opioid receptor family consisting of μ, δ and κ opioid receptors. The anti-opioid properties of its endogenous ligand, N/OFQ provide the receptor interesting potentials in symptoms and processes related to drug addiction, learning and memory, anxiety and depression, and nociception. Using target-selected N-ethyl-N-nitrosourea (ENU)-driven mutagenesis we recently generated a rat model bearing a premature stop codon in the opioid-like receptor (oprl1) gene, and here we describe the primary characterization of this novel model. Data revealed that [3H]N/OFQ binding to brain slices was completely absent in rats homozygous for the premature stop codon (oprl1−/−). Heterozygous rats displayed an intermediate level of NOPr binding. Oprl1 receptor transcript levels, as determined by Northern blot analysis, were reduced by approximately 50% in oprl1−/− rats compared to wild-type controls (oprl1+/+), and no alternative spliced transcripts were observed. Quantitative autoradiographic mapping of μ, δ and κ opioid receptors using [3H]DAMGO, [3H]deltorphin and [3H]CI-977, respectively, did not show any changes in opioid receptor binding. In conclusion, we present a novel mutant rat lacking NOPr without compensatory changes in μ, δ and κ opioid receptors. We anticipate that this mutant rat will have heuristic value to further understand the function of NOPr.

Section snippets

Animals

All experiments were approved by the Animal Care Committee of the Royal Dutch Academy of Sciences and conducted according to the Dutch law for animal experiments. All efforts were made to minimize the amount of animals. NOPr knockout rat (OPRL11Hubr) was generated by target-selected ENU-induced mutagenesis in a Brown Norway background (for detailed description, see Smits et al., 2006). The animals were outcrossed for two generations on a Brown Norway background. Because Brown Norway rats breed

NOPr is completely absent in oprl1−/− rats

High-throughput resequencing of target genes in a library of ENU-mutagenized Brown Norway rats (Smits et al., 2006) revealed a C to G transversion at position 3657 in the oprl1 gene (ENSRNOG00000016768), resulting into a premature stop codon (TAC>TAG) in the third exon. Analysis of [3H]N/OFQ binding revealed that NOPr expression is completely absent in rats homozygous for the oprl1 gene premature stop codon (Fig. 2), indicating that this premature stop codon results into a full knockout of

Discussion

Using recently established gene knockout technology (Smits et al 2006, Smits et al 2008, van Boxtel et al 2008b) we obtained a rat knockout for NOPr. We show that the ENU-induced premature stop codon results into a complete lack of NOPr in homozygous knockout rats. Importantly, no changes were found in μ, δ and κ-opioid receptor binding, arguing that this rat model provides selectiveness towards the inactivation of NOPr and that oprl1−/− rat knockout phenotypes are not likely to be confounded

Conclusion

We conclude that the premature stop codon in the rat oprl1 gene results into a complete knockout of NOPr. The absence of compensatory adaptations in μ, δ and κ opioid receptor expression makes this animal model valuable in the further understanding of NOPr function, its interaction with other neurotransmitter systems, and the assessment of the therapeutic effects of N/OFQ (ant)agonists in pain, Parkinson's disease, and drug addiction (for extensive review see for exampleCiccocioppo et al 2000,

Acknowledgments

We would like to acknowledge Pim Toonen and Mark Verheul for genotyping of animals. This work is funded by the Netherlands Organisation for Scientific Research (NWO), grant # 91676160, awarded to J. R. Homberg and the award “Exploiting natural and induced genetic variation in the laboratory rat” to E.C. from the European Heads of Research Councils and European Science Foundation EURYI (European Young Investigator) Award scheme. NWO, the European Heads of Research Councils, and the European

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