Frédéric Simonin

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.

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.

Decreased Oral Self-Administration of Alcohol In κ-Opioid Receptor Knock-Out Mice

BACKGROUND Although a large body of evidence suggests a role for the opioid system in alcoholism, the precise role of mu-, delta-, kappa-, and ORL1-opioid receptors and the physiological significance of their natural genetic variation have not been identified. The method of targeted gene disruption by homologous recombination has been used to knock out (KO) genes coding for opioid receptors, and study their effects on alcohol self-administration. Here we examined the effects of targeted disruption of kappa-opioid receptor (KOR) on oral alcohol self-administration and other behaviors. METHODS Oral alcohol, saccharin and quinine self-administration was assessed in a two-bottle choice paradigm using escalating concentrations of alcohol, or tastant solutions. In preference tests 12% alcohol, 0.033% and 0.066% saccharin, and 0.03 mM and 0.1 mM quinine solutions were used. Open-field activity was determined in an arena equipped with a computer-controlled activity-detection system. Subjects were tested for three consecutive days. Locomotor activity was assessed on days 1 and 2 (after saline injection, i.p.) and on day 3 (after alcohol injection, i.p.). Alcohol-induced locomotor activity was determined as the difference in activity between day 3 and day 2. RESULTS Male KOR KO mice in preference tests with 12% alcohol consumed about half as much alcohol as wild-type (WT) or heterozygous (HET) mice, showed lower preference for saccharin (0.033% and 0.066%) and higher preference to quinine (0.1 mM) than WT mice. Female KOR KO mice showed similar reduction in alcohol consumption in comparison to WT and HET mice. Partial deletion of KOR in HET mice did not change alcohol consumption in comparison to WT mice. In all genotype-groups females drank significantly more alcohol than males. MANOVA of locomotor activity among KO, WT, and HET mice indicated that strain and sex effects were not significant for alcohol-induced activation (p > 0.05), while strain x sex interaction effects on alcohol-induced activation could be detected (F(1,55) = 6.07, p < 0.05). CONCLUSION Our results indicating decreased alcohol consumption, lower saccharin preference, and higher quinine preference in KOR KO mice are in line with previous observations of opioid involvement in maintenance of food intake and raise the possibility that the deficient dynorphin/KOR system affects orosensory reward through central mechanisms which reduce alcohol intake and disrupt tastant responses, either as direct effects of absence of kappa-opioid receptors, or as effects of indirect developmental compensatory changes.

Identification of a novel family of G protein‐coupled receptor associated sorting proteins

During the past few years several new interacting partners for G protein‐coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein‐coupled receptor associated sorting protein (GASP‐1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP‐1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl‐terminal tail, corresponding to helix 8 in the three‐dimensional structure of rhodopsin. In addition, we show that GASP‐1 interacts with COOH‐terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP‐1. In situ hybridization and northern blot analysis indicate that GASP‐1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP‐1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP‐2, also interacts with the carboxyl‐terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.

Differential involvement of the Mu and Kappa opioid receptors in spatial learning

In order to test the role of mu and kappa opioid receptors (Mu opioid receptor (MOR) and Kappa opioid receptor (KOR)) in hippocampal‐dependent spatial learning, we analyzed genetically engineered null mutant mice missing the functional MOR or KOR gene. Compared to wild‐type mice, the homozygous MOR null mutants exhibited an impairment in the ultimate level of spatial learning as shown in two distinct tasks, the 8‐arm radial‐maze and the Morris water‐maze. Control behaviors were normal. The learning impairment could be associated with the impairment we found in the maintenance of long‐term potentiation in mossy fibers in CA3. In comparison, there was no impairment in spatial learning in our KOR mutants or in mossy fibers (mf) in CA3 region long‐term potentiation (LTP). Our work suggests that the MOR may play a positive role in learning and memory by increasing LTP in CA3 neurons.

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.

Analysis of [3H] bremazocine binding in single and combinatorial opioid receptor knockout mice

Despite ample pharmacological evidence for the existence of multiple mu-, delta- and kappa-opioid receptor subtypes, only three genes encoding mu-(MOR), delta-(DOR) and kappa-(KOR) opioid receptor have been cloned. The KOR gene encodes kappa(1)-sites, which specifically bind arylacetamide compounds, and the possible existence of kappa-opioid receptor subtypes derived from another kappa-opioid-receptor gene, yet to be characterized, remains a very contentious issue. kappa(2)-Opioid receptors are described as binding sites typically labelled by the non-selective benzomorphan ligand [3H]bremazocine in the presence of mu-, delta- and kappa(1)-opioid receptor blocking ligands. To investigate the genetic origin of kappa(2)-opioid receptors, we have carried out homogenate binding experiments with [3H]bremazocine in brains of single MOR-, DOR-, KOR- and double MOR/DOR-deficient mice. Scatchard analysis showed that 68+/-12% of the binding sites arise from the MOR gene, 27+/-1% from the DOR gene and 14.5+/-0.2% from the KOR gene, indicating that the three known genes account for total [3H]bremazocine binding. Experiments in the presence of mu-, delta- and kappa(1)-opioid receptor suppressor ligands further showed that non-kappa(1)-opioid receptor labelling can be accounted for by binding to both the mu- and delta-opioid receptors. Finally, [3H]bremazocine binding experiments performed on brain membranes from the triple MOR/DOR/KOR-deficient mice revealed a complete absence of binding sites, confirming definitively that no additional gene is required to explain the total population of [3H]bremazocine binding sites. Altogether the data show that the putative kappa(2)-opioid receptors are in fact a mixed population of KOR, DOR and predominantly MOR gene products.

Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system.

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies.

Endogenous mammalian RF-amide peptides, including PrRP, kisspeptin and 26RFa, modulate nociception and morphine analgesia via NPFF receptors.

Mammalian RF-amide peptides are encoded by five different genes and act through five different G protein-coupled receptors. RF-amide-related peptides-1 and -3, neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for NPFF1, NPFF2, GPR10, GPR54 and GPR103 receptors, respectively. However, several studies suggest that the selectivity of these peptides for their receptors is low and indicate that expression patterns for receptors and their corresponding ligands only partially overlap. In this study, we took advantage of the cloning of the five human RF-amide receptors to systematically examine their affinity for and their activation by all human RF-amide peptides. Binding experiments, performed on membranes from CHO cells expressing GPR10, GPR54 and GPR103 receptors, confirmed their high affinity and remarkable selectivity for their cognate ligands. Conversely, NPFF1 and NPFF2 receptors displayed high affinity for all RF-amide peptides. Moreover, GTPγS and cAMP experiments showed that almost all RF-amide peptides efficiently activate NPFF1 and NPFF2 receptors. As NPFF is known to modulate morphine analgesia, we undertook a systematic analysis in mice of the hyperalgesic and anti morphine-induced analgesic effects of a representative set of endogenous RF-amide peptides. All of them induced hyperalgesia and/or prevented morphine analgesia following intracerebroventricular administration. Importantly, these effects were prevented by administration of RF9, a highly selective NPFF1/NPFF2 antagonist. Altogether, our results show that all endogenous RF-amide peptides display pain-modulating properties and point to NPFF receptors as essential players for these effects.