A master regulator of opioid reward in the ventral prefrontal cortex.

In addition to their intrinsic rewarding properties, opioids can also evoke aversive reactions that protect against misuse. Cellular mechanisms that govern the interplay between opioid reward and aversion are poorly understood. We used whole-brain activity mapping in mice to show that neurons in the dorsal peduncular nucleus (DPn) are highly responsive to the opioid oxycodone. Connectomic profiling revealed that DPn neurons innervate the parabrachial nucleus (PBn). Spatial and single-nuclei transcriptomics resolved a population of PBn-projecting pyramidal neurons in the DPn that express µ-opioid receptors (µORs). Disrupting µOR signaling in the DPn switched oxycodone from rewarding to aversive and exacerbated the severity of opioid withdrawal. These findings identify the DPn as a key substrate for the abuse liability of opioids.

Distinct µ-opioid ensembles trigger positive and negative fentanyl reinforcement.

Fentanyl is a powerful painkiller that elicits euphoria and positive reinforcement1. Fentanyl also leads to dependence, defined by the aversive withdrawal syndrome, which fuels negative reinforcement2,3 (that is, individuals retake the drug to avoid withdrawal). Positive and negative reinforcement maintain opioid consumption, which leads to addiction in one-fourth of users, the largest fraction for all addictive drugs4. Among the opioid receptors, µ-opioid receptors have a key role5, yet the induction loci of circuit adaptations that eventually lead to addiction remain unknown. Here we injected mice with fentanyl to acutely inhibit γ-aminobutyric acid-expressing neurons in the ventral tegmental area (VTA), causing disinhibition of dopamine neurons, which eventually increased dopamine in the nucleus accumbens. Knockdown of µ-opioid receptors in VTA abolished dopamine transients and positive reinforcement, but withdrawal remained unchanged. We identified neurons expressing µ-opioid receptors in the central amygdala (CeA) whose activity was enhanced during withdrawal. Knockdown of µ-opioid receptors in CeA eliminated aversive symptoms, suggesting that they mediate negative reinforcement. Thus, optogenetic stimulation caused place aversion, and mice readily learned to press a lever to pause optogenetic stimulation of CeA neurons that express µ-opioid receptors. Our study parses the neuronal populations that trigger positive and negative reinforcement in VTA and CeA, respectively. We lay out the circuit organization to develop interventions for reducing fentanyl addiction and facilitating rehabilitation.

Model of negative affect induced by withdrawal from acute and chronic morphine administration in male mice.

Opioid use disorder (OUD) is a chronic relapsing disorder that is a major burden for the lives of affected individuals, and society as a whole. Opioid withdrawal is characterized by strong physical symptoms, along with signs of negative affect. Negative affect due to opioid withdrawal is a major obstacle to recovery and relapse prevention. The mechanisms behind negative affect due to either spontaneous or antagonist-precipitated opioid withdrawal are not well known, and more animal models need be developed. Here, we present behavioral models of negative affect upon naloxone-precipitated morphine withdrawal in adult male mice. Social, anxiety, and despair-like deficits were investigated following naloxone administration in mice receiving morphine under three dosing regimens; acute, chronic constant dose and chronic escalating doses. Social behaviour in the three-chamber social preference test was decreased following withdrawal from chronic and escalating but not acute morphine. Anxiety-like behaviour in the open field was increased for all three treatments. Despair-like behaviour was increased following withdrawal from chronic and escalating but not acute morphine. Altogether, these animal models will contribute to study behavioural and neuronal circuitries involved in the several negative affective signs characterizing OUD.

Manipulating ΔFOSB in D1-Type Medium Spiny Neurons of the Nucleus Accumbens Reshapes Whole-Brain Functional Connectivity.

BACKGROUND: The transcription factor ΔFOSB, acting in the nucleus accumbens, has been shown to control transcriptional and behavioral responses to opioids and other drugs of abuse. However, circuit-level consequences of ΔFOSB induction on the rest of the brain, which are required for its regulation of complex behavior, remain unknown. METHODS: We used an epigenetic approach in mice to suppress or activate the endogenous Fosb gene and thereby decrease or increase, respectively, levels of ΔFOSB selectively in D1-type medium spiny neurons of the nucleus accumbens and tested whether these modifications affect the organization of functional connectivity (FC) in the brain. We acquired functional magnetic resonance imaging data at rest and in response to a morphine challenge and analyzed both stationary and dynamic FC patterns. RESULTS: The 2 manipulations modified brainwide communication markedly and differently. ΔFOSB down- and upregulation had overlapping effects on prefrontal- and retrosplenial cortex-centered networks, but also generated specific FC signatures for epithalamus (habenula) and dopaminergic/serotonergic centers, respectively. Analysis of dynamic FC patterns showed that increasing ΔFOSB essentially altered responsivity to morphine and uncovered striking modifications of the roles of the epithalamus and amygdala in brain communication, particularly upon ΔFOSB downregulation. CONCLUSIONS: These novel findings illustrate how it is possible to link activity of a transcription factor within a single cell type of an identified brain region to consequent changes in circuit function brainwide by use of functional magnetic resonance imaging, and they pave the way for fundamental advances in bridging the gap between transcriptional and brain connectivity mechanisms underlying opioid addiction.

Chronic Morphine Leaves a Durable Fingerprint on Whole-Brain Functional Connectivity.

BACKGROUND: Opioid use disorder is a chronic relapsing disorder. The brain adapts to opioids that are taken for pain treatment or recreational use so that abstinence becomes a true challenge for individuals with opioid use disorder. Studying brain dysfunction at this stage is difficult, and human neuroimaging has provided highly heterogeneous information. METHODS: Here, we took advantage of an established mouse model of morphine abstinence together with functional magnetic resonance imaging to investigate whole-brain functional connectivity (FC) first at rest and then in response to an acute morphine challenge during image acquisition. RESULTS: Hierarchical clustering of seed pair correlation coefficients showed modified FC in abstinent animals, brainwide and regardless of the condition. Seed-to-voxel analysis and random forest classification, performed on data at rest, indicated that the retrosplenial cortex (a core component of the default mode network) and the amygdala (a major aversion center) are the best markers of abstinence, thus validating the translatability of the study. Seed pair network clustering confirmed disruption of a retrosplenial cortex-centered network, reflecting major reorganization of brain FC. The latter analysis also identified a persistent but unreported morphine signature in abstinent mice at rest, which involves cortical and midbrain components and characterizes the enduring morphine footprint. Finally, dynamic FC analysis revealed that the intrascanner acute morphine challenge modified FC faster and more broadly in abstinent animals, demonstrating brainwide adaptations of FC reactivity to an acute opioid challenge. CONCLUSIONS: This study used a unique experimental design to demonstrate that a prior history of chronic opioid exposure leaves a durable pharmacological signature on brain communication, with implications for pain management and recovery from opioid use disorder.

Mu Opioid Receptor-Positive Neurons in the Dorsal Raphe Nucleus Are Impaired by Morphine Abstinence.

BACKGROUND: Chronic opioid exposure leads to hedonic deficits and enhanced vulnerability to addiction, which are observed and even strengthen after a period of abstinence, but the underlying circuit mechanisms are poorly understood. In this study, using both molecular and behavioral approaches, we tested the hypothesis that neurons expressing mu opioid receptors (MORs) in the dorsal raphe nucleus (DRN) are involved in addiction vulnerability associated with morphine abstinence. METHODS: MOR-Cre mice were exposed to chronic morphine and then went through spontaneous withdrawal for 4 weeks, a well-established mouse model of morphine abstinence. We studied DRN-MOR neurons of abstinent mice using 1) viral translating ribosome affinity for transcriptome profiling, 2) fiber photometry to measure neuronal activity, and 3) an opto-intracranial self-stimulation paradigm applied to DRN-MOR neurons to assess responses related to addiction vulnerability including persistence to respond, motivation to obtain the stimulation, self-stimulation despite punishment, and cue-induced reinstatement. RESULTS: DRN-MOR neurons of abstinent animals showed a downregulation of genes involved in ion conductance and MOR-mediated signaling, as well as altered responding to acute morphine. Opto-intracranial self-stimulation data showed that abstinent animals executed more impulsive-like and persistent responses during acquisition and scored higher on addiction-like criteria. CONCLUSIONS: Our data suggest that protracted abstinence to chronic morphine leads to reduced MOR function in DRN-MOR neurons and abnormal self-stimulation of these neurons. We propose that DRN-MOR neurons have partially lost their reward-facilitating properties, which in turn may lead to increased propensity to perform addiction-related behaviors.

Mu Opioid Receptor-Expressing Neurons in the Dorsal Raphe Nucleus Are Involved in Reward Processing and Affective Behaviors.

BACKGROUND: Mu opioid receptors (MORs) are key for reward processing, mostly studied in dopaminergic pathways. MORs are also expressed in the dorsal raphe nucleus (DRN), which is central for the modulation of reward and mood, but MOR function in the DRN remains underexplored. Here, we investigated whether MOR-expressing neurons of the DRN (DRN-MOR neurons) participate in reward and emotional responses. METHODS: We characterized DRN-MOR neurons anatomically using immunohistochemistry and functionally using fiber photometry in responses to morphine and rewarding/aversive stimuli. We tested the effect of opioid uncaging on the DRN on place conditioning. We examined the effect of DRN-MOR neuron optostimulation on positive reinforcement and mood-related behaviors. We mapped their projections and selected DRN-MOR neurons projecting to the lateral hypothalamus for a similar optogenetic experimentation. RESULTS: DRN-MOR neurons form a heterogeneous neuronal population essentially composed of GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons. Calcium activity of DRN-MOR neurons was inhibited by rewarding stimuli and morphine. Local photo-uncaging of oxymorphone in the DRN produced conditioned place preference. DRN-MOR neuron optostimulation triggered real-time place preference and was self-administered, promoted social preference, and reduced anxiety and passive coping. Finally, specific optostimulation of DRN-MOR neurons projecting to the lateral hypothalamus recapitulated the reinforcing effects of total DRN-MOR neuron stimulation. CONCLUSIONS: Our data show that DRN-MOR neurons respond to rewarding stimuli and that their optoactivation has reinforcing effects and promotes positive emotional responses, an activity which is partially mediated by their projections to the lateral hypothalamus. Our study also suggests a complex regulation of DRN activity by MOR opioids, involving mixed inhibition/activation mechanisms that fine-tune DRN function.

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.

Advances in the characterization of negative affect caused by acute and protracted opioid withdrawal using animal models.

Opioid use disorder (OUD) is a chronic brain disease which originates from long-term neuroadaptations that develop after repeated opioid consumption and withdrawal episodes. These neuroadaptations lead among other things to the development of a negative affect, which includes loss of motivation for natural rewards, higher anxiety, social deficits, heightened stress reactivity, an inability to identify and describe emotions, physical and/or emotional pain, malaise, dysphoria, sleep disorders and chronic irritability. The urge for relief from this negative affect is one of major causes of relapse, and thus represents a critical challenge for treatment and relapse prevention. Animal models of negative affect induced by opioid withdrawal have recapitulated the development of a negative emotional state with signs such as anhedonia, increased anxiety responses, increased despair-like behaviour and deficits in social interaction. This research has been critical to determine neurocircuitry adaptations during chronic opioid administration or upon withdrawal. In this review, we summarize the recent literature of rodent models of (i) acute withdrawal, (ii) protracted abstinence from passive administration of opioids, (iii) withdrawal or protracted abstinence from opioid self-administration. Finally, we describe neurocircuitry involved in acute withdrawal and protracted abstinence. This article is part of the Special Issue on "Opioid-induced changes in addiction and pain circuits".

Improvement of the Metabolic Stability of GPR88 Agonist RTI-13951-33: Design, Synthesis, and Biological Evaluation.

GPR88 is an orphan G protein-coupled receptor mainly expressed in the brain, whose endogenous ligand has not yet been identified. To elucidate GPR88 functions, our group has developed RTI-13951-33 (1b) as the first in vivo active GPR88 agonist, but its poor metabolic stability and moderate brain permeability remain to be further optimized. Here, we report the design, synthesis, and pharmacological characterization of a new series of RTI-13951-33 analogues with the aim of improving pharmacokinetic properties. As a result, we identified a highly potent GPR88 agonist RTI-122 (30a) (cAMP EC50 = 11 nM) with good metabolic stability (half-life of 5.8 h) and brain permeability (brain/plasma ratio of >1) in mice. Notably, RTI-122 was more effective than RTI-13951-33 in attenuating the binge-like alcohol drinking behavior in the drinking-in-the-dark paradigm. Collectively, our findings suggest that RTI-122 is a promising lead compound for drug discovery research of GPR88 agonists.

Synthesis and pharmacological validation of a novel radioligand for the orphan GPR88 receptor.

GPR88 is an orphan G protein-coupled receptor which has been implicated in a number of striatal-associated disorders. Herein we describe the synthesis and pharmacological characterization of the first GPR88 radioligand, [3H]RTI-33, derived from a synthetic agonist RTI-13951-33. [3H]RTI-33 has a specific activity of 83.4 Ci/mmol and showed one-site, saturable binding (KD of 85 nM) in membranes prepared from stable PPLS-HA-hGPR88-CHO cells. A competition binding assay was developed to determine binding affinities of several known GPR88 agonists. This radioligand represents a powerful tool for future mechanistic and cell-based ligand-receptor interaction studies of GPR88.

Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

Habenular Neurons Expressing Mu Opioid Receptors Promote Negative Affect in a Projection-Specific Manner.

BACKGROUND: The mu opioid receptor (MOR) is central to hedonic balance and produces euphoria by engaging reward circuits. MOR signaling may also influence aversion centers, notably the habenula (Hb), where the receptor is highly dense. Our previous data suggest that the inhibitory activity of MOR in the Hb may limit aversive states. To investigate this hypothesis, we tested whether neurons expressing MOR in the Hb (Hb-MOR neurons) promote negative affect. METHODS: Using Oprm1-Cre knockin mice, we combined tracing and optogenetics with behavioral testing to investigate consequences of Hb-MOR neuron stimulation for approach/avoidance (real-time place preference), anxiety-related responses (open field, elevated plus maze, and marble burying), and despair-like behavior (tail suspension). RESULTS: Optostimulation of Hb-MOR neurons elicited avoidance behavior, demonstrating that these neurons promote aversive states. Anterograde tracing showed that, in addition to the interpeduncular nucleus, Hb-MOR neurons project to the dorsal raphe nucleus. Optostimulation of Hb-MOR/interpeduncular nucleus terminals triggered avoidance and despair-like responses with no anxiety-related effect, whereas light-activation of Hb-MOR/dorsal raphe nucleus terminals increased levels of anxiety with no effect on other behaviors, revealing 2 dissociable pathways controlling negative affect. CONCLUSIONS: Together, the data demonstrate that Hb neurons expressing MOR facilitate aversive states via 2 distinct Hb circuits, contributing to despair-like behavior (Hb-MOR/interpeduncular nucleus) and anxiety (Hb-MOR/dorsal raphe nucleus). The findings support the notion that inhibition of these neurons by either endogenous or exogenous opioids may relieve negative affect, a mechanism that would have implications for hedonic homeostasis and addiction.

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.

Zn2+ inhibits spatial memory and hippocampal place cell representation through high-affinity binding to the NMDA receptor GluN2A subunit.

A subset of glutamatergic neurons in the forebrain uses labile Zn2+ as a co-transmitter alongside glutamate. Synaptic Zn2+ plays a key role in learning and memory processes, but its mechanisms of action remain poorly understood. Here, we used a knock-in (KI) mouse line carrying a point mutation at the GluN2A Zn2+ binding site that selectively eliminates zinc inhibition of NMDA receptors. Ablation of Zn2+-GluN2A binding improves spatial memory retention and contextual fear memory formation. Electrophysiological recording of hippocampal neurons in the CA1 area revealed a greater proportion of place cells and substantial place field remapping in KI mice compared to wildtype littermates. Persistent place cell remapping was also seen in KI mice upon repeated testing suggesting an enhanced ability to maintain a distinct representation across multiple overlapping experiences. Together, these findings reveal an original molecular mechanism through which synaptic Zn2+ negatively modulates spatial cognition by dampening GluN2A-containing NMDA receptor signaling.

The mu opioid receptor and the orphan receptor GPR151 contribute to social reward in the habenula.

The mu opioid receptor (MOR) and the orphan GPR151 receptor are inhibitory G protein coupled receptors that are enriched in the habenula, a small brain region involved in aversion processing, addiction and mood disorders. While MOR expression in the brain is widespread, GPR151 expression is restricted to the habenula. In a previous report, we created conditional ChrnB4-Cre × Oprm1fl/fl (so-called B4MOR) mice, where MORs are deleted specifically in Chrnb4-positive neurons restricted to the habenula, and shown a role for these receptors in naloxone aversion. Here we characterized the implication of habenular MORs in social behaviors. B4MOR-/- mice and B4MOR+/+ mice were compared in several social behavior measures, including the chronic social stress defeat (CSDS) paradigm, the social preference (SP) test and social conditioned place preference (sCPP). In the CSDS, B4MOR-/- mice showed lower preference for the social target (unfamiliar mouse of a different strain) at baseline, providing a first indication of deficient social interactions in mice lacking habenular MORs. In the SP test, B4MOR-/- mice further showed reduced sociability for an unfamiliar conspecific mouse. In the sCPP, B4MOR-/- mice also showed impaired place preference for their previous familiar littermates after social isolation. We next created and tested Gpr151-/- mice in the SP test, and also found reduced social preference compared to Gpr151+/+ mice. Altogether our results support the underexplored notion that the habenula regulates social behaviors. Also, our data suggest that the inhibitory habenular MOR and GPR151 receptors normally promote social reward, possibly by dampening the aversive habenula activity.

The GPR88 agonist RTI-13951-33 reduces alcohol drinking and seeking in mice.

GPR88 is an orphan G-protein-coupled receptor that is considered a potential target to treat neuropsychiatric disorders, including addiction. Most knowledge about GPR88 function stems from knockout mouse studies, and in vivo pharmacology is still scarce. Here we examine the effects of the novel brain-penetrant agonist RTI-13951-33 on several alcohol-related behaviours in the mouse. In the intermittent-access-two-bottle-choice paradigm, the compound reduced excessive voluntary alcohol drinking, while water drinking was intact. This was observed for C57BL/6 mice, as well as for control but not Gpr88 knockout mice, demonstrating efficacy and specificity of the drug in vivo. In the drinking-in-the-dark paradigm, RTI-13951-33 also reduced binge-like drinking behaviour for control but not Gpr88 knockout mice, confirming the alcohol consumption-reducing effect and in vivo specificity of the drug. When C57BL/6 mice were trained for alcohol self-administration, RTI-13951-33 decreased the number of nose-pokes over a 4-h session and reduced the number of licks and bursts of licks, suggesting reduced motivation to obtain alcohol. Finally, RTI-13951-33 did not induce any place preference or aversion but reduced the expression of conditioned place preference to alcohol, indicative of a reduction of alcohol-reward seeking. Altogether, data show that RTI-13951-33 limits alcohol intake under distinct conditions that require consummatory behaviour, operant response or association with contextual cues. RTI-13951-33 therefore is a promising lead compound to evaluate GPR88 as a therapeutic target for alcohol use disorders. More broadly, RTI-13951-33 represents a unique tool to better understand GPR88 function, disentangle receptor roles in development from those in the adult and perhaps address other neuropsychiatric disorders.

The orphan receptor GPR88 controls impulsivity and is a risk factor for Attention-Deficit/Hyperactivity Disorder.

The neural orphan G protein coupled receptor GPR88 is predominant in the striatum and cortex of both rodents and humans, and considered a potential target for brain disorders. Previous studies have shown multiple behavioral phenotypes in Gpr88 knockout mice, and human genetic studies have reported association with psychosis. Here we tested the possibility that GPR88 contributes to Attention Deficit Hyperactivity Disorder (ADHD). In the mouse, we tested Gpr88 knockout mice in three behavioral paradigms, best translatable between rodents and humans, and found higher motor impulsivity and reduced attention together with the reported hyperactivity. Atomoxetine, a typical ADHD drug, reduced impulsivity in mutant mice. Conditional Gpr88 knockout mice in either D1R-type or D2R-type medium spiny neurons revealed distinct implications of the two receptor populations in waiting and stopping impulsivity. Thus, animal data demonstrate that deficient GPR88 activity causally promotes ADHD-like behaviors, and identify circuit mechanisms underlying GPR88-regulated impulsivity. In humans, we performed a family-based genetic study including 567 nuclear families with DSM-IV diagnosis of ADHD. There was a minor association for SNP rs2036212 with diagnosis, treatment response and cognition. A stronger association was found for SNP rs2809817 upon patient stratification, suggesting that the T allele is a risk factor when prenatal stress is involved. Human data therefore identify GPR88 variants associated with the disease, and highlight a potential role of life trajectories to modulate GPR88 function. Overall, animal and human data concur to suggest that GPR88 signaling should be considered a key factor for diagnostic and treatment of ADHD.