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1.
Neuropsychopharmacology ; 45(2): 247-255, 2020 01.
Article in English | MEDLINE | ID: mdl-31005059

ABSTRACT

The medial habenula (MHb) is considered a brain center regulating aversive states. The mu opioid receptor (MOR) has been traditionally studied at the level of nociceptive and mesolimbic circuits, for key roles in pain relief and reward processing. MOR is also densely expressed in MHb, however, MOR function at this brain site is virtually unknown. Here we tested the hypothesis that MOR in the MHb (MHb-MOR) also regulates aversion processing. We used chnrb4-Cre driver mice to delete the Oprm1 gene in chnrb4-neurons, predominantly expressed in the MHb. Conditional mutant (B4MOR) mice showed habenula-specific reduction of MOR expression, restricted to chnrb4-neurons (50% MHb-MORs). We tested B4MOR mice in behavioral assays to evaluate effects of MOR activation by morphine, and MOR blockade by naloxone. Locomotor, analgesic, rewarding, and motivational effects of morphine were preserved in conditional mutants. In contrast, conditioned place aversion (CPA) elicited by naloxone was reduced in both naïve (high dose) and morphine-dependent (low dose) B4MOR mice. Further, physical signs of withdrawal precipitated by either MOR (naloxone) or nicotinic receptor (mecamylamine) blockade were attenuated. These data suggest that MORs expressed in MHb B4-neurons contribute to aversive effects of naloxone, including negative effect and aversive effects of opioid withdrawal. MORs are inhibitory receptors, therefore we propose that endogenous MOR signaling normally inhibits chnrb4-neurons of the MHb and moderates their known aversive activity, which is unmasked upon receptor blockade. Thus, in addition to facilitating reward at several brain sites, tonic MOR activity may also limit aversion within the MHb circuitry.


Subject(s)
Avoidance Learning/drug effects , Habenula/drug effects , Habenula/metabolism , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Receptors, Opioid, mu/deficiency , Animals , Avoidance Learning/physiology , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Receptors, Opioid, mu/genetics
2.
Science ; 366(6462): 250-254, 2019 10 11.
Article in English | MEDLINE | ID: mdl-31601771

ABSTRACT

The unconventional N-methyl-d-aspartate (NMDA) receptor subunits GluN3A and GluN3B can, when associated with the other glycine-binding subunit GluN1, generate excitatory conductances purely activated by glycine. However, functional GluN1/GluN3 receptors have not been identified in native adult tissues. We discovered that GluN1/GluN3A receptors are operational in neurons of the mouse adult medial habenula (MHb), an epithalamic area controlling aversive physiological states. In the absence of glycinergic neuronal specializations in the MHb, glial cells tuned neuronal activity via GluN1/GluN3A receptors. Reducing GluN1/GluN3A receptor levels in the MHb prevented place-aversion conditioning. Our study extends the physiological and behavioral implications of glycine by demonstrating its control of negatively valued emotional associations via excitatory glycinergic NMDA receptors.


Subject(s)
Behavior, Animal , Emotions , Glycine/metabolism , Habenula/metabolism , Nerve Tissue Proteins/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Calcium/metabolism , Cell Line , Conditioning, Psychological , Cues , Glycine/pharmacology , Humans , Mice , Mice, Knockout , Neuroglia/metabolism , Neurons/metabolism , Patch-Clamp Techniques
3.
eNeuro ; 6(4)2019.
Article in English | MEDLINE | ID: mdl-31346000

ABSTRACT

The orphan receptor GPR88 is highly expressed in D1 receptor (D1R)- and D2R-medium spiny neurons (MSNs) and has been associated to striatum-dependent functions in rodents. The total deletion of Gpr88 in mice was shown to decrease anxiety-like behaviors, increase stereotypies and locomotion, and impair motor coordination and motor learning. Knowing the opposing role of D1R- and D2R-MSNs, we here investigated the respective roles of GPR88 in the two MSN subtypes for these behaviors. To do so, we compared effects of a conditional Gpr88 gene knock-out (KO) in D1R-MSNs (D1R-Gpr88 mice) or D2R-MSNs (A2AR-Gpr88 mice) with effects of the total Gpr88 KO (CMV-Gpr88 mice). Overall, most phenotypes of CMV-Gpr88 mice were recapitulated in A2AR-Gpr88 mice, including reduced marble burying, increased social interactions, increased locomotor activity and stereotypies in the open field, and reduced motor coordination in the rotarod. Exceptions were the reduced habituation to the open field and reduced motor skill learning, which were observed in CMV-Gpr88 and D1R-Gpr88 mice, but not in A2AR-Gpr88 mice. D1R-Gpr88 mice otherwise showed no other phenotype in this study. Our data together show that GPR88 modulates the function of both D1R- and D2R-MSNs, and that GPR88 activity in these two neuron populations has very different and dissociable impacts on behavior. We suggest that GPR88 in D2R-MSNs shapes defensive and social behavior and contributes in maintaining the inhibition of basal ganglia outputs to control locomotion, stereotypies and motor coordination, while GPR88 in D1R-MSNs promotes novelty habituation and motor learning.


Subject(s)
Affect/physiology , Behavior, Animal/physiology , Corpus Striatum/physiology , Neurons/physiology , Receptors, Dopamine D1/physiology , Receptors, Dopamine D2/physiology , Receptors, G-Protein-Coupled/physiology , Animals , Exploratory Behavior/physiology , Female , Male , Mice, Inbred C57BL , Mice, Knockout , Motor Skills/physiology , Social Behavior
4.
Genes Brain Behav ; 17(8): e12473, 2018 11.
Article in English | MEDLINE | ID: mdl-29575471

ABSTRACT

Mouse models are widely used to understand genetic bases of behavior. Traditional testing typically requires multiple experimental settings, captures only snapshots of behavior and involves human intervention. The recent development of automated home cage monitoring offers an alternative method to study mouse behavior in their familiar and social environment, and over weeks. Here, we used the IntelliCage system to test this approach for mouse phenotyping, and studied mice lacking Gpr88 that have been extensively studied using standard testing. We monitored mouse behavior over 22 days in 4 different phases. In the free adaptation phase, Gpr88 -/- mice showed delayed habituation to the home cage, and increased frequency of same corner returns behavior in their alternation pattern. In the following nose-poke adaptation phase, non-habituation continued, however, mutant mice acquired nose-poke conditioning similar to controls. In the place learning and reversal phase, Gpr88-/- mice developed preference for the water/sucrose corner with some delay, but did not differ from controls for reversal. Finally, in a fixed schedule-drinking phase, control animals showed higher activity during the hour preceding water accessibility, and reduced activity after access to water was terminated. Mutant mice did not show this behavior, showing lack of anticipatory behavior. Our data therefore confirm hyperactivity, non-habituation and altered exploratory behaviors that were reported previously. Learning deficits described in other settings were barely detectable, and a novel phenotype was discovered. Home cage monitoring therefore extends previous findings and shows yet another facet of GPR88 function that deserves further investigation.


Subject(s)
Behavior Observation Techniques/instrumentation , Exploratory Behavior/physiology , Receptors, G-Protein-Coupled/metabolism , Animals , Behavior Observation Techniques/methods , Behavior, Animal/physiology , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/genetics , Motor Activity/physiology , Phenotype , Receptors, G-Protein-Coupled/genetics
5.
Mol Psychiatry ; 23(10): 2050-2056, 2018 10.
Article in English | MEDLINE | ID: mdl-29158579

ABSTRACT

Bipolar disorder (BD) is a prevalent mood disorder that tends to cluster in families. Despite high heritability estimates, few genetic susceptibility factors have been identified over decades of genetic research. One possible interpretation for the shortcomings of previous studies to detect causative genes is that BD is caused by highly penetrant rare variants in many genes. We explored this hypothesis by sequencing the exomes of affected individuals from 40 well-characterized multiplex families. We identified rare variants segregating with affected status in many interesting genes, and found an enrichment of deleterious variants in G protein-coupled receptor (GPCR) family genes, which are important drug targets. Furthermore, we showed targeted downstream GPCR dysregulation for some of the variants that may contribute to disease pathology. Particularly interesting was the finding of a rare and functionally relevant nonsense mutation in the corticotropin-releasing hormone receptor 2 (CRHR2) gene that tracked with affected status in one family. By focusing on rare variants in informative families, we identified key biochemical pathways likely implicated in this complex disorder.


Subject(s)
Bipolar Disorder/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Adult , Bipolar Disorder/metabolism , Case-Control Studies , Family , Female , Gene Frequency/genetics , Genetic Linkage , Genetic Predisposition to Disease/genetics , Genetic Variation/genetics , Genome-Wide Association Study , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Middle Aged , Pedigree , Receptors, Corticotropin-Releasing Hormone/genetics , Exome Sequencing
6.
Eur J Neurosci ; 46(4): 2026-2034, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28700108

ABSTRACT

The orphan receptor, GPR88, is emerging as a key player in the pathophysiology of several neuropsychiatric diseases, including psychotic disorders. Knockout (KO) mice lacking GPR88 throughout the brain exhibit many abnormalities relevant to schizophrenia including locomotor hyperactivity, behavioural hypersensitivity to dopaminergic psychostimulants and deficient sensorimotor gating. Here, we used conditional knockout (cKO) mice lacking GPR88 selectively in striatal medium spiny neurons expressing A2A receptor to determine neuronal circuits underlying these phenotypes. We first studied locomotor responses of A2A R-Gpr88 KO mice and their control littermates to psychotomimetic, amphetamine, and to selective D1 and D2 receptor agonists, SKF-81297 and quinpirole, respectively. To assess sensorimotor gating performance, mice were submitted to acoustic and visual prepulse inhibition (PPI) paradigms. Total knockout GPR88 mice were also studied for comparison. Like total GPR88 KO mice, A2A R-Gpr88 KO mice displayed a heightened sensitivity to locomotor stimulant effects of amphetamine and SKF-81297. They also exhibited enhanced locomotor activity to quinpirole, which tended to suppress locomotion in control mice. By contrast, they had normal acoustic and visual PPI, unlike total GPR88 KO mice that show impairments across different sensory modalities. Finally, none of the genetic manipulations altered central auditory temporal processing assessed by gap-PPI. Together, these findings support the role of GPR88 in the pathophysiology of schizophrenia and show that GPR88 in A2A receptor-expressing neurons modulates psychomotor behaviour but not sensorimotor gating.


Subject(s)
Dopamine Agonists/pharmacology , Motor Activity/physiology , Neurons/metabolism , Receptors, Adrenergic, alpha-2/biosynthesis , Receptors, G-Protein-Coupled/biosynthesis , Sensory Gating/physiology , Animals , Female , Gene Expression , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Motor Activity/drug effects , Neurons/drug effects , Receptors, Adrenergic, alpha-2/genetics , Receptors, G-Protein-Coupled/genetics , Reflex, Startle/drug effects , Reflex, Startle/physiology , Sensory Gating/drug effects
7.
Addict Biol ; 22(4): 1010-1021, 2017 Jul.
Article in English | MEDLINE | ID: mdl-27001273

ABSTRACT

Addiction is a chronic brain disorder that progressively invades all aspects of personal life. Accordingly, addiction to opiates severely impairs interpersonal relationships, and the resulting social isolation strongly contributes to the severity and chronicity of the disease. Uncovering new therapeutic strategies that address this aspect of addiction is therefore of great clinical relevance. We recently established a mouse model of heroin addiction in which, following chronic heroin exposure, 'abstinent' mice progressively develop a strong and long-lasting social avoidance phenotype. Here, we explored and compared the efficacy of two pharmacological interventions in this mouse model. Because clinical studies indicate some efficacy of antidepressants on emotional dysfunction associated with addiction, we first used a chronic 4-week treatment with the serotonergic antidepressant fluoxetine, as a reference. In addition, considering prodepressant effects recently associated with kappa opioid receptor signaling, we also investigated the kappa opioid receptor antagonist norbinaltorphimine (norBNI). Finally, we assessed whether fluoxetine and norBNI could reverse abstinence-induced social avoidance after it has established. Altogether, our results show that two interspaced norBNI administrations are sufficient both to prevent and to reverse social impairment in heroin abstinent animals. Therefore, kappa opioid receptor antagonism may represent a useful approach to alleviate social dysfunction in addicted individuals.


Subject(s)
Antidepressive Agents, Second-Generation/therapeutic use , Behavior, Animal/drug effects , Grooming/drug effects , Heroin Dependence/physiopathology , Narcotic Antagonists/therapeutic use , Receptors, Opioid, kappa/antagonists & inhibitors , Animals , Disease Models, Animal , Fluoxetine/therapeutic use , Heroin/pharmacology , Male , Mice , Mice, Inbred C57BL , Naltrexone/analogs & derivatives , Naltrexone/therapeutic use , Social Behavior , Time , Treatment Outcome
8.
Neuroscience ; 313: 46-56, 2016 Jan 28.
Article in English | MEDLINE | ID: mdl-26480813

ABSTRACT

Delta opioid (DOP) receptors participate to the control of chronic pain and emotional responses. Recent data also identified their implication in spatial memory and drug-context associations pointing to a critical role of hippocampal delta receptors. To better appreciate the impact of repeated drug exposure on their modulatory activity, we used fluorescent knock-in mice that express a functional delta receptor fused at its carboxy-terminus with the green fluorescent protein in place of the native receptor. We then tested the impact of chronic morphine treatment on the density and distribution of delta receptor-expressing cells in the hippocampus. A decrease in delta receptor-positive cell density was observed in the CA1, CA3 and dentate gyrus without alteration of the distribution across the different GABAergic populations that mainly express delta receptors. This effect partly persisted after four weeks of morphine abstinence. In addition, we observed increased DOP receptor expression at the cell surface compared to saline-treated animals. In the hippocampus, chronic morphine administration thus induces DOP receptor cellular redistribution and durably decreases delta receptor-expressing cell density. Such modifications are likely to alter hippocampal physiology, and to contribute to long-term cognitive deficits.


Subject(s)
Hippocampus/drug effects , Morphine/pharmacology , Narcotics/pharmacology , Neurons/drug effects , Receptors, Opioid, delta/metabolism , Animals , Chronic Disease , Disease Models, Animal , Female , Gene Knock-In Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hippocampus/metabolism , Hippocampus/pathology , Immunohistochemistry , Male , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Morphine Dependence/metabolism , Morphine Dependence/pathology , Neurons/metabolism , Neurons/pathology , Receptors, Opioid, delta/genetics
9.
Psychopharmacology (Berl) ; 232(11): 1957-71, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25482274

ABSTRACT

RATIONALE: Opiate addiction is a brain disorder emerging through repeated intoxication and withdrawal episodes. Epidemiological studies also indicate that chronic exposure to opiates may lead in susceptible individuals to the emergence of depressive symptoms, strongly contributing to the severity and chronicity of addiction. We recently established a mouse model of heroin abstinence, characterized by the development of depressive-like behaviors following chronic heroin exposure. OBJECTIVES: While genetic factors regulating immediate behavioral responses to opiates have been largely investigated, little is known about their contribution to long-term emotional regulation during abstinence. Here, we compared locomotor stimulation and physical dependence induced by heroin exposure, as well as emotional dysfunction following abstinence, across mice strains with distinct genetic backgrounds. METHODS: Mice from three inbred strains (C57BL/6J, Balb/cByJ, and 129S2/SvPas) were exposed to an escalating chronic heroin regimen (10-50 mg/kg). Independent cohorts were used to assess drug-induced locomotor activity during chronic treatment, naloxone-precipitated withdrawal at the end of chronic treatment, and emotional-like responses after a 4-week abstinence period. RESULTS: Distinct behavioral profiles were observed across strains during heroin treatment, with no physical dependence and low locomotor stimulation in 129S2/SvPas. In addition, different behavioral impairments developed during abstinence across the three strains, with increased despair-like behavior in 129S2/SvPas and Balb/cByJ, and low sociability in 129S2/SvPas and C57BL/6J. CONCLUSIONS: Our results indicate that depressive-like behaviors emerge during heroin abstinence, whatever the severity of immediate behavioral responses to the drug. In addition, inbred mouse strains will allow studying several aspects of mood-related deficits associated with addiction.


Subject(s)
Arousal/drug effects , Arousal/genetics , Emotions/drug effects , Heroin Dependence/genetics , Heroin Dependence/psychology , Heroin/pharmacology , Motor Activity/drug effects , Motor Activity/genetics , Animals , Heroin/toxicity , Male , Mice , Mice, Inbred Strains , Naloxone/pharmacology , Social Behavior , Substance Withdrawal Syndrome/genetics , Substance Withdrawal Syndrome/psychology
10.
Neuroscience ; 277: 595-609, 2014 Sep 26.
Article in English | MEDLINE | ID: mdl-25086313

ABSTRACT

The habenular complex, encompassing medial (MHb) and lateral (LHb) divisions, is a highly conserved epithalamic structure involved in the dorsal diencephalic conduction system (DDC). These brain nuclei regulate information flow between the limbic forebrain and the mid- and hindbrain, integrating cognitive with emotional and sensory processes. The MHb is also one of the strongest expression sites for mu opioid receptors (MORs), which mediate analgesic and rewarding properties of opiates. At present however, anatomical distribution and function of these receptors have been poorly studied in MHb pathways. Here we took advantage of a newly generated MOR-mcherry knock-in mouse line to characterize MOR expression sites in the DDC. MOR-mcherry fluorescent signal is weak in the LHb, but strong expression is visible in the MHb, fasciculus retroflexus (fr) and interpeduncular nucleus (IPN), indicating that MOR is mainly present in the MHb-IPN pathway. MOR-mcherry cell bodies are detected both in basolateral and apical parts of MHb, where the receptor co-localizes with cholinergic and substance P (SP) neurons, respectively, representing two main MHb neuronal populations. MOR-mcherry is expressed in most MHb-SP neurons, and is present in only a subpopulation of MHb-cholinergic neurons. Intense diffuse fluorescence detected in lateral and rostral parts of the IPN further suggests that MOR-mcherry is transported to terminals of these SP and cholinergic neurons. Finally, MOR-mcherry is present in septal regions projecting to the MHb, and in neurons of the central and intermediate IPN. Together, this study describes MOR expression in several compartments of the MHb-IPN circuitry. The remarkably high MOR density in the MHb-IPN pathway suggests that these receptors are in a unique position to mediate analgesic, autonomic and reward responses.


Subject(s)
Habenula/metabolism , Interpeduncular Nucleus/metabolism , Receptors, Opioid, mu/metabolism , Acetylcholine/metabolism , Animals , Enkephalins/metabolism , Female , Gene Knock-In Techniques , Immunohistochemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Neural Pathways/metabolism , Neurons/metabolism , Protein Precursors/metabolism , Receptors, Opioid, mu/genetics , Substance P/metabolism , Red Fluorescent Protein
11.
Br J Pharmacol ; 171(23): 5376-86, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25048778

ABSTRACT

BACKGROUND AND PURPOSE: Activation of δ opioid (DOP) receptors regulates pain and emotional responses, and also displays ligand-biased agonism. KNT-127 (1,2,3,4,4a,5,12,12a-octahydro-2-methyl-4aß,1ß-([1,2]benzenomethano)-2,6-diazanaphthacene-12aß,17-diol) is a novel DOP receptor agonist inducing analgesia and antidepressant effects in mice. Here, we have assessed KNT-127 for (i) analgesia against chronic inflammatory pain; (ii) effects on depression, locomotion and DOP receptor internalization; and (iii) for cross-tolerance to analgesic and antidepressant effects of acute treatment by other DOP receptor agonists. EXPERIMENTAL APPROACH: Inflammatory pain was induced by complete Freund's adjuvant injection into tail or hindpaw, and thermal and mechanical sensitivities were determined in mice. Locomotor and antidepressant-like effects were measured using actimetry and forced swim test respectively. In vivo KNT-127 selectivity and internalization were assessed using DOP receptor knockout mice and knock-in mice expressing fluorescent-tagged DOP receptors. KNT-127 was injected acutely at 0.1-10.0 mg·kg(-1) or administered chronically at 5 mg·kg(-1) daily over 5 days. KEY RESULTS: Acute treatment with KNT-127 reversed inflammatory hyperalgesia, produced an antidepressant-like effect but induced neither hyperlocomotion nor receptor sequestration. Chronic treatment with KNT-127 induced tolerance and cross-tolerance to SNC80-induced analgesia, but no tolerance to SNC80-evoked hyperlocomotor or antidepressant-like effects. CONCLUSIONS AND IMPLICATIONS: The DOP receptor agonist KNT-127 induced agonist-specific acute and chronic responses, at both behavioural and cellular levels. It displays activities similar to the other recently reported DOP agonists, AR-M1000390, ADL5747 and ADL5859, and differs from SNC80. SNC80 differs from the other DOP receptor agonists including KNT-127, by exhibiting ligand-biased tolerance at this receptor.


Subject(s)
Analgesics/therapeutic use , Antidepressive Agents/therapeutic use , Depression/drug therapy , Morphinans/therapeutic use , Pain/drug therapy , Receptors, Opioid, delta/agonists , Analgesics/pharmacology , Animals , Antidepressive Agents/pharmacology , Behavior, Animal/drug effects , Benzamides/pharmacology , Drug Tolerance , Freund's Adjuvant , Hot Temperature , Hyperalgesia/drug therapy , Male , Mice, Inbred C57BL , Mice, Knockout , Morphinans/pharmacology , Motor Activity/drug effects , Pain/etiology , Piperazines/pharmacology , Receptors, Opioid, delta/genetics , Receptors, Opioid, delta/metabolism
12.
Behav Brain Res ; 243: 44-52, 2013 Apr 15.
Article in English | MEDLINE | ID: mdl-23295400

ABSTRACT

A crucial issue in treating opiate addiction, a chronic relapsing disorder, is to maintain a drug-free abstinent state. Prolonged abstinence associates with mood disorders, strongly contributing to relapse. In particular, substance use disorders occurring during adolescence predispose to depression later in adulthood. Using our established mouse model of opiate abstinence, we characterized emotional consequences into adulthood of morphine exposure during adolescence. Our results indicate that morphine treatment in adolescent mice has no effect on anxiety-like behaviours in adult mice, after abstinence. In contrast, morphine treatment during adolescence increases behavioural despair in adult mice. We also show that morphine exposure strain-dependently enhances sociability in adult mice. Additional research will be required to understand where and how morphine acts during brain maturation to affect emotional and social behaviours into adulthood.


Subject(s)
Aging/drug effects , Anxiety/drug therapy , Behavior, Animal/drug effects , Emotions/drug effects , Morphine/pharmacology , Narcotics/pharmacology , Social Behavior , Aging/physiology , Animals , Anxiety/chemically induced , Chronic Disease , Disease Models, Animal , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Morphine/administration & dosage , Morphine Dependence/drug therapy , Morphine Dependence/physiopathology , Morphine Dependence/psychology , Narcotics/administration & dosage , Neuropsychological Tests , Substance Withdrawal Syndrome/drug therapy
13.
Neuroscience ; 221: 203-13, 2012 Sep 27.
Article in English | MEDLINE | ID: mdl-22750239

ABSTRACT

Delta opioid receptors participate to the control of chronic pain and emotional responses. Recent data also identified their implication in spatial memory and drug-context associations pointing to a critical role of hippocampal delta receptors. We examined the distribution of delta receptor-expressing cells in the hippocampus using fluorescent knock-in mice that express a functional delta receptor fused at its carboxyterminus with the green fluorescent protein in place of the native receptor. Colocalization with markers for different neuronal populations was performed by immunohistochemical detection. Fine mapping in the dorsal hippocampus confirmed that delta opioid receptors are mainly present in GABAergic neurons. Indeed, they are mostly expressed in parvalbumin-immunopositive neurons both in the Ammon's horn and dentate gyrus. These receptors, therefore, most likely participate in the dynamic regulation of hippocampal activity.


Subject(s)
Hippocampus/cytology , Neurons/metabolism , Receptors, Opioid, delta/metabolism , Animals , Female , Glutamate Decarboxylase/metabolism , Green Fluorescent Proteins/genetics , Hippocampus/anatomy & histology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Parvalbumins/metabolism , Receptors, Opioid, delta/genetics , Somatostatin/metabolism , Vesicular Glutamate Transport Protein 1/metabolism , Vesicular Glutamate Transport Protein 2/metabolism
14.
Neuroscience ; 218: 185-95, 2012 Aug 30.
Article in English | MEDLINE | ID: mdl-22613736

ABSTRACT

Cholecystokinin (CCK) is a neuropeptide widely distributed in the mammalian brain. This peptide regulates many physiological functions and behaviors, such as cardio-respiratory control, thermoregulation, nociception, feeding, memory processes and motivational responses, and plays a prominent role in emotional responses including anxiety and depression. CCK-expressing brain regions involved in these functions remain unclear and their identification represents an important step towards understanding CCK function in the brain. The basolateral amygdala (BLA) is strongly involved in emotional processing and expresses high levels of CCK. In this study we examined the contribution of CCK expressed in this brain region to emotional responses in mice. To knockdown CCK specifically in the BLA, we used stereotaxic delivery of recombinant adeno-associated viral vectors expressing a CCK-targeted shRNA. This procedure efficiently reduced CCK levels locally. shCCK-treated animals showed reduced levels of anxiety in the elevated plus-maze, and lower despair-like behavior in the forced swim test. Our data demonstrate that CCK expressed in the BLA represents a key brain substrate for anxiogenic and depressant effects of the peptide. The study also suggests that elevated amygdalar CCK could contribute to panic and major depressive disorders that have been associated with CCK dysfunction in humans.


Subject(s)
Amygdala/metabolism , Anxiety/metabolism , Cholecystokinin/metabolism , Depression/metabolism , Animals , Cholecystokinin/deficiency , Gene Knockdown Techniques , Image Processing, Computer-Assisted , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , Reverse Transcriptase Polymerase Chain Reaction
15.
Nucleic Acids Res ; 38(21): 7749-63, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20675720

ABSTRACT

7SK snRNA, an abundant RNA discovered in human nucleus, regulates transcription by RNA polymerase II (RNAPII). It sequesters and inhibits the transcription elongation factor P-TEFb which, by phosphorylation of RNAPII, switches transcription from initiation to processive elongation and relieves pauses of transcription. This regulation process depends on the association between 7SK and a HEXIM protein, neither isolated partner being able to inhibit P-TEFb alone. In this work, we used a combined NMR and biochemical approach to determine 7SK and HEXIM1 elements that define their binding properties. Our results demonstrate that a repeated GAUC motif located in the upper part of a hairpin on the 5'-end of 7SK is essential for specific HEXIM1 recognition. Binding of a peptide comprising the HEXIM Arginine Rich Motif (ARM) induces an opening of the GAUC motif and stabilization of an internal loop. A conserved proline-serine sequence in the middle of the ARM is shown to be essential for the binding specificity and the conformational change of the RNA. This work provides evidences for a recognition mechanism involving a first event of induced fit, suggesting that 7SK plasticity is involved in the transcription regulation.


Subject(s)
RNA, Small Nuclear/chemistry , RNA-Binding Proteins/metabolism , Base Pairing , Base Sequence , Binding Sites , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , RNA, Small Nuclear/metabolism , RNA-Binding Proteins/chemistry , Repetitive Sequences, Nucleic Acid , Transcription Factors
16.
Neuroscience ; 156(4): 950-65, 2008 Oct 28.
Article in English | MEDLINE | ID: mdl-18786617

ABSTRACT

The central extended amygdala (EAc) is an ensemble of highly interconnected limbic structures of the anterior brain, and forms a cellular continuum including the bed nucleus of the stria terminalis (BNST), the central nucleus of the amygdala (CeA) and the nucleus accumbens shell (AcbSh). This neural network is a key site for interactions between brain reward and stress systems, and has been implicated in several aspects of drug abuse. In order to increase our understanding of EAc function at the molecular level, we undertook a genome-wide screen (Affymetrix) to identify genes whose expression is enriched in the mouse EAc. We focused on the less-well known BNST-CeA areas of the EAc, and identified 121 genes that exhibit more than twofold higher expression level in the EAc compared with whole brain. Among these, 43 genes have never been described to be expressed in the EAc. We mapped these genes throughout the brain, using non-radioactive in situ hybridization, and identified eight genes with a unique and distinct rostro-caudal expression pattern along AcbSh, BNST and CeA. Q-PCR analysis performed in brain and peripheral organ tissues indicated that, with the exception of one (Spata13), all these genes are predominantly expressed in brain. These genes encode signaling proteins (Adora2, GPR88, Arpp21 and Rem2), a transcription factor (Limh6) or proteins of unknown function (Rik130, Spata13 and Wfs1). The identification of genes with enriched expression expands our knowledge of EAc at a molecular level, and provides useful information to toward genetic manipulations within the EAc.


Subject(s)
Amygdala/physiology , Gene Expression Profiling/methods , Gene Expression/physiology , Nerve Tissue Proteins/genetics , Amygdala/anatomy & histology , Animals , Brain Mapping , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Oligonucleotide Array Sequence Analysis
17.
Eur J Neurosci ; 27(11): 2973-84, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18588537

ABSTRACT

Addiction develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. The central extended amygdala (EAc) is a network formed by the central amygdala and the bed nucleus of the stria terminalis. This key site controls drug craving and seeking behaviors, and has not been investigated at the gene regulation level. We used Affymetrix microarrays to analyze transcriptional activity in the murine EAc, with a focus on mu-opioid receptor-associated events because these receptors mediate drug reward and dependence. We identified 132 genes whose expression is regulated by a chronic escalating morphine regimen in the EAc from wild-type but not mu-opioid receptor knockout mice. These modifications are mostly EAc-specific. Gene ontology analysis reveals an overrepresentation of neurogenesis, cell growth and signaling protein categories. A separate quantitative PCR analysis of genes in the last of these groups confirms the dysregulation of both orphan (Gpr88) and known (DrD1A, Adora2A, Cnr1, Grm5, Gpr6) G protein-coupled receptors, scaffolding (PSD95, Homer1) and signaling (Sgk, Cap1) proteins, and neuropeptides (CCK, galanin). These transcriptional modifications do not occur following a single morphine injection, and hence result from long-term adaptation to excessive mu receptor activation. Proteins encoded by these genes are classically associated with spine modules function in other brain areas, and therefore our data suggest a remodeling of EAc circuits at sites where glutamatergic and monoaminergic afferences interact. Together, mu receptor-dependent genes identified in this study potentially contribute to drug-induced neural plasticity, and provide a unique molecular repertoire towards understanding drug craving and relapse.


Subject(s)
Amygdala/drug effects , Analgesics, Opioid/pharmacology , Gene Expression Regulation/drug effects , Neuronal Plasticity/drug effects , Receptors, Opioid, mu/agonists , Transcriptional Activation/drug effects , Amygdala/metabolism , Animals , Gene Expression Regulation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Morphine/pharmacology , Neural Pathways/drug effects , Neural Pathways/metabolism , Neuronal Plasticity/genetics , Oligonucleotide Array Sequence Analysis , Opioid-Related Disorders/genetics , Opioid-Related Disorders/metabolism , Opioid-Related Disorders/physiopathology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, Opioid, mu/metabolism , Septal Nuclei/drug effects , Septal Nuclei/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Transcriptional Activation/genetics
18.
Ann N Y Acad Sci ; 1129: 175-84, 2008.
Article in English | MEDLINE | ID: mdl-18591478

ABSTRACT

The lateral hypothalamus (LH) is a brain structure that controls hedonic properties of both natural rewards and drugs of abuse. Mu opioid receptors are known to mediate drug reward, but whether overstimulation of these receptors impacts on LH function has not been studied. Here we have used a genome-wide microarray approach to identify LH responses to chronic mu opioid receptor activation at the transcriptional level. We have subjected wild-type and mu opioid receptor knockout mice to an escalating morphine regimen, which produces severe physical dependence in wild-type but not mutant animals. We have analyzed gene profiles in LH samples using the 430A.2 Affymetrix array and identified a set of 25 genes whose expression is altered by morphine in wild-type mice only. The regulation was confirmed for a subset of these genes using real-time quantitative PCR on samples from independent treatments. Altered expression of aquaporin 4, apolipoprotein D, and prostaglandin synthase is indicative of modified LH physiology. The regulation of two signaling genes (the serum glucocorticoid kinase and the regulator of G protein signaling 4) suggests that neurotransmission is altered in LH circuitry. Finally, the downregulation of apelin may indicate a potential role for this neuropeptide in opioid signaling and hedonic homeostasis. Altogether, our study shows that chronic mu opioid receptor stimulation induces gene expression plasticity in the LH and provides a unique collection of mu opioid receptor-dependent genes that potentially contribute to alter reward processes in addictive diseases.


Subject(s)
Gene Expression Regulation , Hypothalamus/metabolism , Receptors, Opioid, mu/genetics , Animals , Gene Expression Regulation/drug effects , Hypothalamus/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Morphine/pharmacology , Polymerase Chain Reaction , Receptors, Opioid, mu/metabolism , Reproducibility of Results
19.
J Neurochem ; 105(3): 865-73, 2008 May.
Article in English | MEDLINE | ID: mdl-18182056

ABSTRACT

Mu and delta opioid receptors (MORs and DORs) were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Galpha(i/o) protein in human embryonic kidney 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Galpha(i/o) proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N,N(CH3)2-Dmt-Tic-NH2. When a MOR fused to a non-functional Galpha subunit was co-expressed with the DOR-Galpha protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced DOR coupling to its tethered Galpha subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the DOR and also results in activation of the Galpha subunit fused the DOR.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Opioid, delta/metabolism , Receptors, Opioid, mu/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , Analgesics, Opioid/pharmacology , Cell Line , Central Nervous System/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Humans , Narcotic Antagonists/pharmacology , Neurons/metabolism , Nociceptors/metabolism , Opioid Peptides/metabolism , Pain/genetics , Pain/metabolism , Pain/physiopathology , Protein Binding/physiology , Protein Conformation , Receptors, G-Protein-Coupled/genetics , Receptors, Opioid, delta/drug effects , Receptors, Opioid, delta/genetics , Receptors, Opioid, mu/drug effects , Receptors, Opioid, mu/genetics , Recombinant Fusion Proteins/genetics
20.
Neuroscience ; 146(3): 1182-92, 2007 May 25.
Article in English | MEDLINE | ID: mdl-17395390

ABSTRACT

Large-scale transcriptome analysis in the brain is a powerful approach to identify novel genes of potential interest toward understanding cerebral organization and function. We utilized the microarray technology to measure expression levels of about 24,000 genes and expressed sequence tags in mouse hippocampus, frontal cortex and striatum. Using expression profile obtained from whole brain as a reference, we categorized the genes into groups of genes either enriched in, or restricted to, one of the three areas of interest. We found enriched genes for each target area. Further, we identified 14 genes in the category of genes restricted to the striatum, among which were the orphan G protein-coupled receptor GPR88 and retinoic acid receptor-beta. These two genes were already reported to be selectively expressed in the striatum, thus validating our experimental approach. We selected 6 striatal-restricted genes, as well as 10 striatal-enriched candidates, that were previously undescribed. We analyzed their expression by in situ hybridization analysis in the brain, and quantitative RT-PCR in both brain and peripheral organs. Two of these unknown genes displayed a notable expression pattern. The striatal-restricted gene H3076B11 shows uniform expression throughout and uniquely in the striatum, representing a genuine striatal marker. The striatal-enriched gene 4833421E05Rik is preferentially expressed in the rostral striatum, and is also abundant in kidney, liver and lung. These two genes may contribute to some of the many striatal-controlled behaviors, including initiation of movement, habit formation, or reward and motivation.


Subject(s)
Brain Chemistry/genetics , Gene Expression Profiling , Neostriatum/metabolism , Nerve Tissue Proteins/biosynthesis , Animals , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Data Interpretation, Statistical , In Situ Hybridization , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Oligonucleotide Array Sequence Analysis , RNA/biosynthesis , RNA/genetics , Receptors, Retinoic Acid/biosynthesis , Receptors, Retinoic Acid/genetics , Reverse Transcriptase Polymerase Chain Reaction
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