168 research outputs found
Enhanced spontaneous activity of the mu opioid receptor by cysteine mutations: characterization of a tool for inverse agonist screening.
BACKGROUND: The concept of spontaneous- or constitutive-activity has become widely accepted and verified for numerous G protein-coupled receptors and this ligand-independent activity is also acknowledged to play a role in some pathologies. Constitutive activity has been reported for the mu opioid receptor. In some cases the increase in receptor basal activity was induced by chronic morphine administration suggesting that constitutive activity may contribute to the development of drug tolerance and dependence. Constitutively active mutants represent excellent tools for gathering information about the mechanisms of receptor activation and the possible physiological relevance of spontaneous receptor activity. The high basal level of activity of these mutants also allows for easier identification of inverse agonists, defined as ligands able to suppress spontaneous receptor activity, and leads to a better comprehension of their modulatory effects as well as possible in vivo use. RESULTS: Cysteines 348 and 353 of the human mu opioid receptor (hMOR) were mutated into alanines and Ala(348,353 )hMOR was stably expressed in HEK 293 cells. [(35)S] GTPγS binding experiments revealed that Ala(348,353 )hMOR basal activity was significantly higher when compared to hMOR, suggesting that the mutant receptor is constitutively active. [(35)S] GTPγS binding was decreased by cyprodime or CTOP indicating that both ligands have inverse agonist properties. All tested agonists exhibited binding affinities higher for Ala(348,353 )hMOR than for hMOR, with the exception of endogenous opioid peptides. Antagonist affinity remained virtually unchanged except for CTOP and cyprodime that bound the double mutant with higher affinities. The agonists DAMGO and morphine showed enhanced potency for the Ala(348,353 )hMOR receptor in [(35)S] GTPγS experiments. Finally, pretreatment with the antagonists naloxone, cyprodime or CTOP significantly increased Ala(348,353 )hMOR expression. CONCLUSION: Taken together our data indicate that the double C348/353A mutation results in a constitutively active conformation of hMOR that is still activated by agonists. This is the first report of a stable CAM of hMOR with the potential to screen for inverse agonists
Mu and Delta Opioid Receptors Oppositely Regulate Motor Impulsivity in the Signaled Nose Poke Task
Impulsivity is a primary feature of many psychiatric disorders, most notably attention deficit hyperactivity disorder and drug addiction. Impulsivity includes a number of processes such as the inability to delay gratification, the inability to withhold a motor response, or acting before all of the relevant information is available. These processes are mediated by neural systems that include dopamine, serotonin, norepinephrine, glutamate and cannabinoids. We examine, for the first time, the role of opioid systems in impulsivity by testing whether inactivation of the mu- (Oprm1) or delta- (Oprd1) opioid receptor gene alters motor impulsivity in mice. Wild-type and knockout mice were examined on either a pure C57BL6/J (BL6) or a hybrid 50% C57Bl/6J–50% 129Sv/pas (HYB) background. Mice were trained to respond for sucrose in a signaled nose poke task that provides independent measures of associative learning (responses to the reward-paired cue) and motor impulsivity (premature responses). Oprm1 knockout mice displayed a remarkable decrease in motor impulsivity. This was observed on the two genetic backgrounds and did not result from impaired associative learning, as responses to the cue signaling reward did not differ across genotypes. Furthermore, mutant mice were insensitive to the effects of ethanol, which increased disinhibition and decreased conditioned responding in wild-type mice. In sharp contrast, mice lacking the Oprd1 gene were more impulsive than controls. Again, mutant animals showed no deficit in associative learning. Ethanol completely disrupted performance in these animals. Together, our results suggest that mu-opioid receptors enhance, whereas delta-opioid receptors inhibit, motor impulsivity. This reveals an unanticipated contribution of endogenous opioid receptor activity to disinhibition. In a broader context, these data suggest that alterations in mu- or delta-opioid receptor function may contribute to impulse control disorders
In vivo neuronal co-expression of mu and delta opioid receptors uncovers new therapeutic perspectives
Opioid receptors are G protein coupled receptors that modulate brain function at all levels of neural integration, including autonomous, sensory, emotional and cognitive processing. Mu and delta opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular level remains unsolved. Also, the notion of receptor crosstalk via mu-delta heteromers is well documented in vitro but in vivo evidence remains scarce. To identify neurons in which receptor interactions could take place, we designed a unique double mutant knock-in mouse line that expresses functional red-fluorescent mu receptors and green-fluorescent delta receptors. We mapped mu and delta receptor distribution and co-localization throughout the nervous system and created the first interactive brain atlas with concomitant mu-delta visualization at subcellular resolution (http://mordor.ics-mci.fr/). Mu and delta receptors co-localize in neurons from subcortical networks but are mainly detected in separate neurons in the forebrain. Also, co-immunoprecipitation experiments indicated physical proximity in the hippocampus, a prerequisite to mu-delta heteromerization. Altogether, data suggest that mu-delta functional interactions take place at systems level for high-order emotional and cognitive processing whereas mu-delta may interact at cellular level in brain networks essential for survival, which has potential implications for innovative drug design in pain control, drug addiction and eating disorders
Circadian-Dependent Learning and Memory Enhancement in Nociceptin Receptor-Deficient Mice with a Novel KUROBOX Apparatus Using Stress-Free Positive Cue Task
Delta opioid receptors regulate temporoammonic-activated feedforward inhibition to the mouse CA1 hippocampus
The opioid system influences learning and memory processes. However, neural mechanisms underlying the modulation of hippocampal activity by opioid receptors remain largely unknown. Here, we compared how mu and delta receptors operate within the mouse CA1 network, and used knock-in mice expressing functional delta opioid receptors fused to the green fluorescent protein (DOR-eGFP) to determine how delta opioid receptor-expressing interneurons integrate within the hippocampal circuitry. Through whole cell patch-clamp recording of CA1 pyramidal neurons from wild-type and DOR-eGFP mice, we found that mu and delta receptors both modulate spontaneous GABAergic inhibition received by these cells. Interestingly, mu but not delta receptor activation decreased the feed-forward inhibitory input evoked by Schaffer collateral stimulation. However, mu and delta agonists modulated GABAergic feed-forward inhibition when evoked upon stimulation of the temporoammonic pathway. In addition, anterograde tracing using biotinylated dextran amine injected into the entorhinal cortex of DOR-eGFP mice suggests the existence of synaptic contacts between temporoammonic afferents and delta receptor-expressing interneurons processes in CA1. Altogether, our data demonstrate a distinct modulatory role of the hippocampal network activity by mu and delta opioid receptors, and show for the first time that delta receptor-expressing interneurons in the CA1 are recruited by the temporoammonic pathway rather than the Schaffer collateral.Funding: The authors thank their funding sources including the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université de Strasbourg, The Agence Nationale pour la Recherche (IMOP), the National Institutes of Health (NIDA DA-05010) and the Stefan and Shirley Hatos Center for europharmacology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Anti-inflammatory properties of the μ opioid receptor support its use in the treatment of colon inflammation
Kappa Opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons
The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR(−/−) mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release. SIGNIFICANCE STATEMENT Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood. Understanding how KOR activation produces dysphoria is key to the development of better analgesics and to defining how the endogenous dynorphin opioids produce their depression-like effects. Results in this study show that the aversive effects of κ receptor activation required arrestin-dependent p38α MAPK activation in dopamine neurons but did not require inhibition of dopamine release in the nucleus accumbens. Thus, contrary to the prevailing view, inhibition of mesolimbic dopamine release does not mediate the aversive effects of KOR activation and functionally selective κ opioids that do not activate arrestin signaling may be effective analgesics lacking dysphoric effects
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