Deformation-based Morphometry MRI Reveals Brain Structural Modifications in Living Mu Opioid Receptor Knockout Mice

Mu opioid receptor (MOR) activation facilitates reward processing and reduces pain, and brain networks underlying these effects are under intense investigation. Mice lacking the MOR gene (MOR KO mice) show lower drug and social reward, enhanced pain sensitivity and altered emotional responses. Our previous neuroimaging analysis using Resting-state (Rs) functional Magnetic Resonance Imaging (fMRI) showed significant alterations of functional connectivity (FC) within reward/aversion networks in these mice, in agreement with their behavioral deficits. Here we further used a structural MRI approach to determine whether volumetric alterations also occur in MOR KO mice. We acquired anatomical images using a 7-Tesla MRI scanner and measured deformation-based morphometry (DBM) for each voxel in subjects from MOR KO and control groups. Our analysis shows marked anatomical differences in mutant animals. We observed both local volumetric contraction (striatum, nucleus accumbens, bed nucleus of the stria terminalis, hippocampus, hypothalamus and periacqueducal gray) and expansion (prefrontal cortex, amygdala, habenula, and periacqueducal gray) at voxel level. Volumetric modifications occurred mainly in MOR-enriched regions and across reward/aversion centers, consistent with our prior FC findings. Specifically, several regions with volume differences corresponded to components showing highest FC changes in our previous Rs-fMRI study, suggesting a possible function-structure relationship in MOR KO-related brain differences. In conclusion, both Rs-fMRI and volumetric MRI in live MOR KO mice concur to disclose functional and structural whole-brain level mechanisms that likely drive MOR-controlled behaviors in animals, and may translate to MOR-associated endophenotypes or disease in humans

Réponses à la morphine in vivo (adaptations moléculaires et implications de la kinase RSK2)

La toxicomanie est une pathologie chronique et récidivante, caractérisée par une recherche compulsive de drogue, une perte de contrôle de la consommation et une très forte probabilité de rechute. La morphine est à la fois une drogue toxicomanogène et un médicament utilisé pour lutter contre des douleurs sévères. L action pharmacologique de la morphine est médiée par le récepteur aux opioïdes mu. Au sein du laboratoire, nous étudions les adaptations moléculaires et comportementales qui se développent suite à l activation chronique du récepteur mu. Mon travail de thèse porte sur les régulations géniques et la signalisation intracellulaire associées à l activation du récepteur mu in vivo.Une première partie de mon travail de thèse a porté sur les adaptations transcriptionnelles consécutives à l activation chronique du récepteur mu in vivo. Nous nous sommes focalisés sur les régulations de l expression des gènes dans deux structures du cerveau encore peu étudiées et impliquées dans les aspects émotionnels de l addiction (amygdale étendue centrale et hypothalamus latéral) et nous avons opté pour une stratégie à l échelle du génome. Dans une deuxième série d expériences, j ai contribué à une caractérisation moléculaire de l état d abstinence.Dans la deuxième partie de ma thèse, j ai étudié la contribution de la kinase RSK2 dans les réponses comportementales à la morphine in vivo. Cette kinase, potentiellement effectrice de l activation du récepteur mu, n a jusqu à présent pas été étudiée dans le cadre de la toxicomanie. Nous avons étudié des souris knockout pour le gène RSK2 dans plusieurs tests permettant d évaluer les effets de la morphine, en administration aigue ou chronique. Nos résultats suggèrent un rôle de la kinase RSK2 dans l analgésie à la morphine et le sevrage.Drug addiction is a chronic disorder characterized by compulsive drug seeking, a loss of control over drug consumption and an important risk of relapse. Morphine is used to treat pain, and is also a drug of abuse. Morphine acts via the mu opioid receptor. In our laboratory, we are studying molecular and behavioral adaptations developing after chronic activation of the mu receptor. The aim of this thesis was to study the regulation of gene expression and intracellular pathways associated with activation of the mu receptor in vivo.A first part of my thesis addressed the transcriptional adaptations consecutive to chronic activation of the mu receptor in vivo. We used a genome-wide microarray approach to study modifications of gene expression in two brain structures (central extended amygdala and lateral hypothalamus). These brain areas have been poorly studied in the context of drug abuse, and are known to be involved in the emotional aspects of addiction. In a second set of experiments, I contributed to the molecular characterization of an abstinent state.In the second part of my thesis, I studied the implication of RSK2 kinase in behavioral responses to morphine since, a role that had never been investigated before. In order to examine the implication of RSK2 in both acute and adaptative responses to morphine, we compared several morphine effects in RSK2 deficient mice and wild-type controls. We tested morphine analgesia and tolerance, morphine locomotor sensitization, morphine physical dependence and morphine reward. Our data reveal a role of RSK2 in morphine analgesia and withdrawal.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

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

GPCR and Alcohol-Related Behaviors in Genetically Modified Mice

International audienceG protein-coupled receptors (GPCRs) constitute the largest class of cell surface signaling receptors and regulate major neurobiological processes. Accordingly, GPCRs represent primary targets for the treatment of brain disorders. Several human genetic polymorphisms affecting GPCRs have been associated to different components of alcohol use disorder (AUD). Moreover, GPCRs have been reported to contribute to several features of alcohol-related behaviors in animal models. Besides traditional pharmacological tools, genetic-based approaches mostly aimed at deleting GPCR genes provided substantial information on how key GPCRs drive alcohol-related behaviors. In this review, we summarize the alcohol phenotypes that ensue from genetic manipulation, in particular gene deletion, of key GPCRs in rodents. We focused on GPCRs that belong to fundamental neuronal systems that have been shown as potential targets for the development of AUD treatment. Data are reviewed with particular emphasis on alcohol reward, seeking, and consumption which are behaviors that capture essential aspects of AUD. Literature survey indicates that in most cases, there is still a gap in defining the intracellular transducers and the functional crosstalk of GPCRs as well as the neuronal populations in which their signaling regulates alcohol actions. Further, the implication of only a few orphan GPCRs has been so far investigated in animal models. Combining advanced pharmacological technologies with more specific genetically modified animals and behavioral preclinical models is likely necessary to deepen our understanding in how GPCR signaling contributes to AUD and for drug discovery

Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum.

The tyrosine kinase Fyn plays an important role in synaptic plasticity, learning, and memory. Here we report that Fyn is activated in response to 15 min D1 receptor (D1R) but not D2 receptor (D2R) stimulation specifically in the dorsomedial striatum (DMS) of mice but not in the other substriatal regions, the dorsolateral striatum (DLS), and the nucleus accumbens (NAc). Once activated Fyn phosphorylates its substrate GluN2B, and we show that GluN2B is phosphorylated only in the DMS but not in the other striatal regions. Striatal neurons are divided into D1R expressing medium spiny neurons (MSNs) and D2R expressing MSNs. Thus, to explore the cell-type specificity of this signaling pathway in the DMS, we developed a Cre-dependent Flip Excision (FLEX) approach to knockdown Fyn in D1R MSNs or D2R MSNs, and proved that the D1R-dependent Fyn activation is localized to DMS D1R MSNs. Importantly, we provide evidence to suggest that the differential association of Fyn and GluN2B with the scaffolding RACK1 is due to the differential localization of Fyn in lipid rafts.Our data further suggest that the differential cholesterol content in the three striatal regions may determine the region specificity of this signaling pathway. Together, our data show that the D1R-dependent Fyn/GluN2B pathway is selectively activated in D1R expressing MSNs in the DMS, and that the brain region specificity of pathway depends on the molecular and cellular compartmentalization of Fyn and GluN2B

Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

The tyrosine kinase Fyn plays an important role in synaptic plasticity, learning, and memory. Here we report that Fyn is activated in response to 15 min D1 receptor (D1R) but not D2 receptor (D2R) stimulation specifically in the dorsomedial striatum (DMS) of mice but not in the other substriatal regions, the dorsolateral striatum (DLS), and the nucleus accumbens (NAc). Once activated Fyn phosphorylates its substrate GluN2B, and we show that GluN2B is phosphorylated only in the DMS but not in the other striatal regions. Striatal neurons are divided into D1R expressing medium spiny neurons (MSNs) and D2R expressing MSNs. Thus, to explore the cell-type specificity of this signaling pathway in the DMS, we developed a Cre-dependent Flip Excision (FLEX) approach to knockdown Fyn in D1R MSNs or D2R MSNs, and proved that the D1R-dependent Fyn activation is localized to DMS D1R MSNs. Importantly, we provide evidence to suggest that the differential association of Fyn and GluN2B with the scaffolding RACK1 is due to the differential localization of Fyn in lipid rafts.Our data further suggest that the differential cholesterol content in the three striatal regions may determine the region specificity of this signaling pathway. Together, our data show that the D1R-dependent Fyn/GluN2B pathway is selectively activated in D1R expressing MSNs in the DMS, and that the brain region specificity of pathway depends on the molecular and cellular compartmentalization of Fyn and GluN2B

Striatal-enriched protein tyrosine phosphatase controls responses to aversive stimuli: implication for ethanol drinking.

The STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific phosphatase whose dysregulation in expression and/or activity is associated with several neuropsychiatric disorders. We recently showed that long-term excessive consumption of ethanol induces a sustained inhibition of STEP activity in the dorsomedial striatum (DMS) of mice. We further showed that down-regulation of STEP expression in the DMS, and not in the adjacent dorsolateral striatum, increases ethanol intake, suggesting that the inactivation of STEP in the DMS contributes to the development of ethanol drinking behaviors. Here, we compared the consequence of global deletion of the STEP gene on voluntary ethanol intake to the consumption of an appetitive rewarding substance (saccharin) or an aversive solution (quinine or denatonium). Whereas saccharin intake was similar in STEP knockout (KO) and wild type (WT) littermate mice, the consumption of ethanol as well as quinine and denatonium was increased in STEP KO mice. These results suggested that the aversive taste of these substances was masked upon deletion of the STEP gene. We therefore hypothesized that STEP contributes to the physiological avoidance towards aversive stimuli. To further test this hypothesis, we measured the responses of STEP KO and WT mice to lithium-induced conditioned place aversion (CPA) and found that whereas WT mice developed lithium place aversion, STEP KO mice did not. In contrast, conditioned place preference (CPP) to ethanol was similar in both genotypes. Together, our results indicate that STEP contributes, at least in part, to the protection against the ingestion of aversive agents