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.

Targeting Morphine-Responsive Neurons: Generation of a Knock-In Mouse Line Expressing Cre Recombinase from the Mu-Opioid Receptor Gene Locus.

The mu-opioid receptor (MOR) modulates nociceptive pathways and reward processing, and mediates the strong analgesic and addictive properties of both medicinal as well as abused opioid drugs. MOR function has been extensively studied, and tools to manipulate or visualize the receptor protein are available. However, circuit mechanisms underlying MOR-mediated effects are less known, because genetic access to MOR-expressing neurons is lacking. Here we report the generation of a knock-in Oprm1-Cre mouse line, which allows targeting and manipulating MOR opioid-responsive neurons. A cDNA encoding a T2A cleavable peptide and Cre recombinase fused to enhanced green fluorescent protein (EGFP/Cre) was inserted downstream of the Oprm1 gene sequence. The resulting Oprm1-Cre line shows intact Oprm1 gene transcription. MOR and EGFP/Cre proteins are coexpressed in the same neurons, and localized in cytoplasmic and nuclear compartments, respectively. MOR signaling is unaltered, demonstrated by maintained DAMGO-induced G-protein activation, and in vivo MOR function is preserved as indicated by normal morphine-induced analgesia, hyperlocomotion, and sensitization. The Cre recombinase efficiently drives the expression of Cre-dependent reporter genes, shown by local virally mediated expression in the medial habenula and brain-wide fluorescence on breeding with tdTomato reporter mice, the latter showing a distribution patterns typical of MOR expression. Finally, we demonstrate that optogenetic activation of MOR neurons in the ventral tegmental area of Oprm1-Cre mice evokes strong avoidance behavior, as anticipated from the literature. The Oprm1-Cre line is therefore an excellent tool for both mapping and functional studies of MOR-positive neurons, and will be of broad interest for opioid, pain, and addiction research.

The Negative Affect of Protracted Opioid Abstinence: Progress and Perspectives From Rodent Models.

Opioid use disorder (OUD) is characterized by the development of a negative emotional state that develops after a history of long-term exposure to opioids. OUD represents a true challenge for treatment and relapse prevention. Human research has amply documented emotional disruption in individuals with an opioid substance use disorder, at both behavioral and brain activity levels; however, brain mechanisms underlying this particular facet of OUD are only partially understood. Animal research has been instrumental in elucidating genes and circuits that adapt to long-term opioid use or are modified by acute withdrawal, but research on long-term consequences of opioid exposure and their relevance to the negative affect of OUD remains scarce. In this article, we review the literature with a focus on two questions: 1) Do we have behavioral models in rodents, and what do they tell us? and 2) What do we know about the neuronal populations involved? Behavioral rodent models have successfully recapitulated behavioral signs of the OUD-related negative affect, and several neurotransmitter systems were identified (i.e., serotonin, dynorphin, corticotropin-releasing factor, oxytocin). Circuit mechanisms driving the negative mood of prolonged abstinence likely involve the 5 main reward-aversion brain centers (i.e., nucleus accumbens, bed nucleus of the stria terminalis, amygdala, habenula, and raphe nucleus), all of which express mu opioid receptors and directly respond to opioids. Future work will identify the nature of these mu opioid receptor-expressing neurons throughout reward-aversion networks, characterize their adapted phenotype in opioid abstinent animals, and hopefully position these primary events in the broader picture of mu opioid receptor-associated brain aversion networks.

"If walls could talk": A photo-elicitation-based observation of service users' perceptions of the care setting and of its influence on the therapeutic alliance in addiction treatment.

A good quality therapeutic alliance is central to the support and treatment of people who use psychoactive substances. Although previous research has suggested that place has an important role in sustaining the therapeutic alliance, this issue has been insufficiently explored in the field of addiction treatment. We conducted a qualitative study using photo-elicitation and interviewing service users in an outpatient addiction treatment centre. They reported both strongly positive and negative perceptions of the place, alongside an unstable therapeutic alliance. Apprehending the place in which care is delivered as a dynamic relational network helps to understand the role of place in shaping the therapeutic alliance in addiction treatment. There is a need for careful design and layout, and thoughtful organisation of these places.

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.

Causal role for the subthalamic nucleus in interrupting behavior.

Stopping or pausing in response to threats, conflicting information, or surprise is fundamental to behavior. Evidence across species has shown that the subthalamic nucleus (STN) is activated by scenarios involving stopping or pausing, yet evidence that the STN causally implements stops or pauses is lacking. Here we used optogenetics to activate or inhibit mouse STN to test its putative causal role. We first demonstrated that optogenetic stimulation of the STN excited its major projection targets. Next we showed that brief activation of STN projection neurons was sufficient to interrupt or pause a self-initiated bout of licking. Finally, we developed an assay in which surprise was used to interrupt licking, and showed that STN inhibition reduced the disruptive effect of surprise. Thus STN activation interrupts behavior, and blocking the STN blunts the interruptive effect of surprise. These results provide strong evidence that the STN is both necessary and sufficient for such forms of behavioral response suppression.

A proteomic and transcriptomic view of amino acids catabolism in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica has to develop dynamic metabolic adaptation mechanisms to survive within the cheese habitat. The availability of amino acids (AAs) is of major importance for microbial development and/or aroma production during cheese ripening. Using 2-D protein gel electrophoresis, we analyzed the adaptation mechanisms of Y. lipolytica for AAs limitation or supplementation in a batch culture containing lactate as a carbon source. Proteome analyses allow the identification of 34 differentially expressed proteins between the culture conditions. These analyses demonstrated that prior to the AAs addition, mainly proteins involved in the oxidative stress of the yeast were induced. Following the AAs addition, yeast cells reorganize their metabolism toward AAs catabolism and also generate a higher induction of proteins related to carbon metabolism and proteins biosynthesis. Using real-time reverse transcription PCR, we re-evaluated the expression of genes encoding proteins involved in these processes. The expression levels of the genes were in accordance with the proteomic results, with the up-regulation of genes encoding a branched-chain amino transferase BAT2, a pyruvate decarboxylase PDC6 and an Hsp70 protein SSZ1 involved in protein biosynthesis. A volatile compound analysis was also performed, and increased production of dimethyldisulfide from methionine and 3-methyl-butanal from leucine was observed in media supplemented with AAs.

Soil eukaryotic functional diversity, a metatranscriptomic approach.

To appreciate the functional diversity of communities of soil eukaryotic micro-organisms we evaluated an experimental approach based on the construction and screening of a cDNA library using polyadenylated mRNA extracted from a forest soil. Such a library contains genes that are expressed by each of the different organisms forming the community and represents its metatranscriptome. The diversity of the organisms that contributed to this library was evaluated by sequencing a portion of the 18S rDNA gene amplified from either soil DNA or reverse-transcribed RNA. More than 70% of the sequences were from fungi and unicellular eukaryotes (protists) while the other most represented group was the metazoa. Calculation of richness estimators suggested that more than 180 species could be present in the soil samples studied. Sequencing of 119 cDNA identified genes with no homologues in databases (32%) and genes coding proteins involved in different biochemical and cellular processes. Surprisingly, the taxonomic distribution of the cDNA and of the 18S rDNA genes did not coincide, with a marked under-representation of the protists among the cDNA. Specific genes from such an environmental cDNA library could be isolated by expression in a heterologous microbial host, Saccharomyces cerevisiae. This is illustrated by the functional complementation of a histidine auxotrophic yeast mutant by two cDNA originating possibly from an ascomycete and a basidiomycete fungal species. Study of the metatranscriptome has the potential to uncover adaptations of whole microbial communities to local environmental conditions. It also gives access to an abundant source of genes of biotechnological interest.

How does a symbiotic fungus modulate expression of its host-plant nitrite reductase?

* In the mycorrhizal association, changes in the metabolic activities expressed by the plant and fungal partners could result from modulations in the quantity and nature of nutrients available at the plant-fungus interface. This hypothesis was tested for the nitrite reductase gene in the association Hebeloma cylindrosporumxPinus pinaster. * Transcripts from plant and fungal nitrite reductases and a fungal ammonium transporter were quantified in control uninoculated roots, extraradical mycelia and mycorrhizas formed by either wild-type or nitrate reductase deficient fungal strains. * The fungal genes were downregulated in mycorrhizas compared with extraradical hyphae. The plant nitrite reductase was induced only transiently by NO(3)(-) in the association with a wild-type strain, but permanently expressed at a high level in mycorrhizas formed by the deficient mutant. * These results suggest that reduced nitrogen compounds transferred from the fungus to the root cortical cells repress the plant nitrite reductase, thus highlighting a plant gene regulation by the nutrients available in the Hartig net.