Hans W.D Matthes

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.

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.

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.

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.

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.

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.

Orphanin FQ/nociceptin binds to functionally coupled ORL1 receptors on human immune cell lines and alters peripheral blood mononuclear cell proliferation.

Orphanin FQ/nociceptin (OFQ/N) has been shown to modulate nociception, responses to stress and anxiety. We investigated OFQ/N function in human immune cells. We find that monocytic U937, T lymphocytic CEM, and MOLT-4 cell lines express OFQ/N binding sites at levels comparable to that of human SH-SY5Y neuroblastoma cells. We show that OFQ/N receptors are functionally coupled to G proteins in these cells. Finally OFQ/N decreases proliferation of phytohemagglutinin-stimulated peripheral blood mononuclear cells in vitro at doses ranging from 10(-13) to 10(-8) M. Thus, our data suggest that OFQ/N and OFQ/N receptor may act as an immunomodulatory system.

Specific activation of the μ opioid receptor (MOR) by endomorphin 1 and endomorphin 2

The recently discovered endomorphin 1 (Tyr‐Pro‐Trp‐Phe‐NH2) and endomorphin 2 (Tyr‐Pro‐Phe‐Phe‐NH2) were investigated with respect to their direct receptor‐binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the μ opioid receptor, an effect reversed by the μ receptor antagonist CTAP (d‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2), but they had no influence on cells stably expressing the δ opioid receptor. To further establish the selectivity of these peptides for the μ opioid receptor, brain preparations of mice lacking the μ opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98u2003TBq/mmol (53.4u2003Ci/mmol), labelled the brain membranes of wild‐type mice with a Kd value of 1.77u2003nm and a Bmax of 63.33u2003fmol/mg protein. In membranes of mice lacking the μreceptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine‐5′‐O‐(3‐thio)triphosphate ([35S]GTPγS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the μ opioid receptors.

Lack of dependence and rewarding effects of deltorphin II in mu-opioid receptor-deficient mice

We have previously shown that the antinociceptive effects produced by the delta opioid‐selective agonist deltorphin II are preserved in mu‐opioid receptor (MOR)‐deficient mice. We have now investigated rewarding effects and physical dependence produced by deltorphin II in these animals. Wild‐type and MOR‐deficient mice were implanted with a cannula into the third ventricle and deltorphin II was administered centrally. The rewarding effects induced by deltorphin II were then investigated using the place preference paradigm. Wild‐type mice showed place preference for the compartment previously associated with deltorphin II and this effect was not observed in MOR‐deficient mice. In a second experiment, mice received a chronic perfusion of deltorphin II over 6u2003days, via an Alzet minipump connected to the intraventricular cannula, and withdrawal was precipitated by naloxone administration. Wild‐type animals showed a moderate but significant incidence of several somatic signs of withdrawal. This withdrawal response was suppressed in MOR‐deficient mice. Analysis of the immunoreactivity levels of PKC‐alpha, PKC‐beta (I and II) and PKC‐gamma isozymes in the cerebral cortex of mice infused chronically with deltorphin II showed a significant up‐regulation of all these isozymes in the soluble fraction in wild‐type but not in MOR‐deficient mice. In conclusion, mu‐opioid receptors, which are not involved in deltorphin II antinociception, appear to mediate the effects of chronic deltorphin II on rewarding responses, physical dependence and adaptive changes to PKC.