Selective targeting of mu opioid receptors to primary cilia.

Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both in vivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.

Chronic tianeptine induces tolerance in analgesia and hyperlocomotion via mu-opioid receptor activation in mice.

Introduction: Tianeptine is approved in some countries to treat depression and anxiety. In addition to its activity on serotonin and glutamate neurotransmission, tianeptine has been proven to be a mu-opioid receptor (MOR) agonist, but only a few preclinical studies have characterized the opioid-like behavioral effects of tianeptine. Methods: In this study, we tested tianeptine activity on G protein activation using the [S35] GTPγS binding assay in brain tissue from MOR+/+ and MOR-/- mice. Then, to determine whether tianeptine behavioral responses are MOR-dependent, we characterized the analgesic, locomotor, and rewarding responses of tianeptine in MOR+/+ and MOR-/- mice using tail immersion, hot plate, locomotor, and conditioned place preference tests. Results: Using the [S35] GTPγS binding assay, we found that tianeptine signaling is mediated by MOR in the brain with properties similar to those of DAMGO (a classic MOR agonist). Furthermore, we found that the MOR is necessary for tianeptine's analgesic (tail immersion and hot plate), locomotor, and rewarding (conditioned place preference) effects. Indeed, these behavioral effects could only be measured in MOR+/+ mice but not in MOR-/- mice. Additionally, chronic administration of tianeptine induced tolerance to its analgesic and hyperlocomotor effects. Discussion: These findings suggest that tianeptine's opioid-like effects require MOR and that chronic use could lead to tolerance.

Distinct and sex-specific expression of mu opioid receptors in anterior cingulate and somatosensory S1 cortical areas.

ABSTRACT: The anterior cingulate cortex (ACC) processes the affective component of pain, whereas the primary somatosensory cortex (S1) is involved in its sensory-discriminative component. Injection of morphine in the ACC has been reported to be analgesic, and endogenous opioids in this area are required for pain relief. Mu opioid receptors (MORs) are expressed in both ACC and S1; however, the identity of MOR-expressing cortical neurons remains unknown. Using the Oprm1-mCherry mouse line, we performed selective patch clamp recordings of MOR+ neurons, as well as immunohistochemistry with validated neuronal markers, to determine the identity and laminar distribution of MOR+ neurons in ACC and S1. We found that the electrophysiological signatures of MOR+ neurons differ significantly between these 2 areas, with interneuron-like firing patterns more frequent in ACC. While MOR+ somatostatin interneurons are more prominent in ACC, MOR+ excitatory neurons and MOR+ parvalbumin interneurons are more prominent in S1. Our results suggest a differential contribution of MOR-mediated modulation to ACC and S1 outputs. We also found that females had a greater density of MOR+ neurons compared with males in both areas. In summary, we conclude that MOR-dependent opioidergic signaling in the cortex displays sexual dimorphisms and likely evolved to meet the distinct function of pain-processing circuits in limbic and sensory cortical areas.

Visualization of real-time receptor endocytosis in dopamine neurons enabled by NTSR1-Venus knock-in mice.

Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental area (VTA). A subset of these neurons expresses the neurotensin receptor NTSR1 and its putative ligand neurotensin (Nts). NTSR1, a G protein-coupled receptor (GPCR), which classically activates Gαq/calcium signaling, is a potential route for modulating DA activity. Drug development efforts have been hampered by the receptor's complex pharmacology and a lack of understanding about its endogenous location and signaling responses. Therefore, we have generated NTSR1-Venus knock-in (KI) mice to study NTSR1 receptors in their physiological context. In primary hippocampal neurons, we show that these animals express functional receptors that respond to agonists by increasing intracellular calcium release and trafficking to endosomes. Moreover, systemic agonist administration attenuates locomotion in KIs as it does in control animals. Mapping receptor protein expression at regional and cellular levels, located NTSR1-Venus on the soma and dendrites of dopaminergic SN/VTA neurons. Direct monitoring of receptor endocytosis, as a proxy for activation, enabled profiling of NTSR1 agonists in neurons, as well as acute SN/VTA containing brain slices. Taken together, NTSR1-Venus animals express traceable receptors that will improve understanding of NTSR1 and DA activities and more broadly how GPCRs act in vivo.

Ackr3-Venus knock-in mouse lights up brain vasculature.

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits ßarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

Recent advances in basic science methodology to evaluate opioid safety profiles and to understand opioid activities.

Opioids are powerful drugs used by humans for centuries to relieve pain and are still frequently used as pain treatment in current clinical practice. Medicinal opioids primarily target the mu opioid receptor (MOR), and MOR activation produces unmatched pain-alleviating properties, as well as side effects such as strong rewarding effects, and thus abuse potential, and respiratory depression contributing to death during overdose. Therefore, the ultimate goal is to create opioid pain-relievers with reduced respiratory depression and thus fewer chances of lethality. Efforts are also underway to reduce the euphoric effects of opioids and avoid abuse liability. In this review, recent advances in basic science methodology used to understand MOR pharmacology and activities will be summarized. The focus of the review will be to describe current technological advances that enable the study of opioid analgesics from subcellular mechanisms to mesoscale network responses. These advances in understanding MOR physiological responses will help to improve knowledge and future design of opioid analgesics.

Biased Signaling of the Mu Opioid Receptor Revealed in Native Neurons.

G protein-coupled receptors are key signaling molecules and major targets for pharmaceuticals. The concept of ligand-dependent biased signaling raises the possibility of developing drugs with improved efficacy and safety profiles, yet translating this concept to native tissues remains a major challenge. Whether drug activity profiling in recombinant cell-based assays, traditionally used for drug discovery, has any relevance to physiology is unknown. Here we focused on the mu opioid receptor, the unrivalled target for pain treatment and also the key driver for the current opioid crisis. We selected a set of clinical and novel mu agonists, and profiled their activities in transfected cell assays using advanced biosensors and in native neurons from knock-in mice expressing traceable receptors endogenously. Our data identify Gi-biased agonists, including buprenorphine, and further show highly correlated drug activities in the two otherwise very distinct experimental systems, supporting in vivo translatability of biased signaling for mu opioid drugs.

Current strategies toward safer mu opioid receptor drugs for pain management.

INTRODUCTION: Pain relief remains a major public health challenge. The most efficient available painkillers are opioids targeting the mu opioid receptor (MOR). MORs are expressed in the areas of the brain [including pain and respiratory centers] that are important for processing reward and aversion. Thus, MOR activation efficiently alleviates severe pain, but the concomitant reward and respiratory depressant effects pose a threat; patients taking opioids potentially develop opioid addiction and high risk for overdose. Areas covered: Ongoing efforts to generate safer opioid analgesics are reviewed here. The design of biased compounds that trigger MOR induced G protein over ß-arrestin signaling, peripheral opioids, drugs targeting MORs in heteromers and drugs enhancing endogenous opioid activity are discussed. Expert opinion: There is evidence that throttling MOR signaling may lead to an era of opioids that are truly efficient painkillers with lower side effects and risk of overdose. However, few of the drugs derived from the advanced approaches outlined here, are getting approval by regulatory committees for use in clinical settings. Thus, there is an urgent need to (i) better clarify mechanisms underlying the hazardous physiological effects of MOR activation, and (ii) fully validate the safety of these new MOR-based therapies.

Oxycodone-Mediated Activation of the Mu Opioid Receptor Reduces Whole Brain Functional Connectivity in Mice.

Oxycodone is a potent medicinal opioid analgesic to treat pain. It is also addictive and a main cause for the current opioid crisis. At present, the impact of oxycodone on coordinated brain network activities, and contribution of the mu opioid receptor (MOR) to these effects, is unknown. We used pharmacological magnetic resonance imaging in mice to characterize MOR-mediated oxycodone effects on whole-brain functional connectivity (FC). Control (CTL) and MOR knockout (KO) animals were imaged under dexmedetomidine in a 7Tesla scanner. Acquisition was performed continuously before and after 2 mg/kg oxycodone administration (analgesic in CTL mice). Independent component analysis (data-driven) produced a correlation matrix, showing widespread oxycodone-induced reduction of FC across 71 components. Isocortex, nucleus accumbens (NAc), pontine reticular nucleus, and periacqueducal gray (PAG) components showed the highest number of significant changes. Seed-to-voxel FC analysis (hypothesis-driven) was then focused on PAG and NAc considered key pain and reward centers. The two seeds showed reduced FC with 8 and 22 Allen Brain Atlas-based regions, respectively, in CTL but not KO mice. Further seed-to-seed quantification showed highest FC modifications of both PAG and NAc seeds with hypothalamic and amygdalar areas, as well as between them, revealing the strongest impact across reward and aversion/pain centers of the brain. In conclusion, we demonstrate that oxycodone reduces brain communication in a MOR-dependent manner, and establish a preliminary whole-brain FC signature of oxycodone. This proof-of-principle study provides a unique platform and reference data set to test other MOR opioid agonists and perhaps discover new mechanisms and FC biomarkers predicting safer analgesics.

Expression map of 78 brain-expressed mouse orphan GPCRs provides a translational resource for neuropsychiatric research.

Orphan G-protein-coupled receptors (oGPCRs) possess untapped potential for drug discovery. In the brain, oGPCRs are generally expressed at low abundance and their function is understudied. Expression profiling is an essential step to position oGPCRs in brain function and disease, however public databases provide only partial information. Here, we fine-map expression of 78 brain-oGPCRs in the mouse, using customized probes in both standard and supersensitive in situ hybridization. Images are available at http://ogpcr-neuromap.douglas.qc.ca. This searchable database contains over 8000 coronal brain sections across 1350 slides, providing the first public mapping resource dedicated to oGPCRs. Analysis with public mouse (60 oGPCRs) and human (56 oGPCRs) genome-wide datasets identifies 25 oGPCRs with potential to address emotional and/or cognitive dimensions of psychiatric conditions. We probe their expression in postmortem human brains using nanoString, and included data in the resource. Correlating human with mouse datasets reveals excellent suitability of mouse models for oGPCRs in neuropsychiatric research.

Mapping GPR88-Venus illuminates a novel role for GPR88 in sensory processing.

GPR88 is an orphan G-protein coupled receptor originally characterized as a striatal-enriched transcript and is a potential target for neuropsychiatric disorders. At present, gene knockout studies in the mouse have essentially focused on striatal-related functions and a comprehensive knowledge of GPR88 protein distribution and function in the brain is still lacking. Here, we first created Gpr88-Venus knock-in mice expressing a functional fluorescent receptor to fine-map GPR88 localization in the brain. The receptor protein was detected in neuronal soma, fibers and primary cilia depending on the brain region, and remarkably, whole-brain mapping revealed a yet unreported layer-4 cortical lamination pattern specifically in sensory processing areas. The unique GPR88 barrel pattern in L4 of the somatosensory cortex appeared 3 days after birth and persisted into adulthood, suggesting a potential function for GPR88 in sensory integration. We next examined Gpr88 knockout mice for cortical structure and behavioral responses in sensory tasks. Magnetic resonance imaging of live mice revealed abnormally high fractional anisotropy, predominant in somatosensory cortex and caudate putamen, indicating significant microstructural alterations in these GPR88-enriched areas. Further, behavioral analysis showed delayed responses in somatosensory-, visual- and olfactory-dependent tasks, demonstrating a role for GPR88 in the integration rather than perception of sensory stimuli. In conclusion, our data show for the first time a prominent role for GPR88 in multisensory processing. Because sensory integration is disrupted in many psychiatric diseases, our study definitely positions GPR88 as a target to treat mental disorders perhaps via activity on cortical sensory networks.

Prostaglandin E receptor EP1 forms a complex with dopamine D1 receptor and directs D1-induced cAMP production to adenylyl cyclase 7 through mobilizing G(ßγ) subunits in human embryonic kidney 293T cells.

The mechanism underlying the crosstalk between multiple G protein-coupled receptors remains poorly understood. We previously reported that prostaglandin E receptor EP1 facilitates dopamine D1 receptor signaling in striatal slices and promotes behavioral responses induced by D1 receptor agonists. Here, using human embryonic kidney (HEK)-293T cells expressing D1 and EP1, we have analyzed the mechanism underlying EP1-mediated facilitation of D1 receptor signaling. Fluorescent immunostaining showed that EP1 and D1 receptors are partly colocalized in the cells, and coprecipitation experiments revealed a molecular complex of EP1 and D1 receptors. Treatment of the cells with 17S,17,20-dimethyl-2,5-ethano-6-oxo-PGE1 (ONO-DI-004), an EP1-selective agonist, enhanced cAMP production induced by D1 agonists (±)-6-chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide (SKF-81297) and 6-chloro-2,3,4,5-tetrahydro-1-(3-methylphenyl)-3-(2-propenyl)-1H-3-benzazepine-7,8-diol hydrobromide (SKF-83822). Although this facilitative effect of EP1 stimulation was not affected by pharmacologic blockade of EP1-induced Ca²âº increase, it was blocked by overexpression of G(tα) as a G(ßγ) scavenger. Consistently, depletion of adenylyl cyclase (AC) 7, a G(ßγ)-sensitive AC isoform, abolished the facilitative action of EP1 on D1-induced cAMP production. Notably, neither G(tα) overexpression nor AC7 depletion affected cAMP production induced by D1 stimulation alone. In contrast, depletion of AC6, another AC isoform, reduced cAMP production induced by D1 stimulation alone, but spared its facilitation by EP1 stimulation. Collectively, these data suggest that, through complex formation with D1, EP1 signaling directs the D1 receptor through G(ßγ) to be coupled to AC7, an AC isoform distinct from those used by the D1 receptor alone, in HEK-293T cells.