Astroglial Knockout of Glucocorticoid Receptor Attenuates Morphine Withdrawal Symptoms, but Not Antinociception and Tolerance in Mice.

The development of tolerance and drug dependence limit the clinical application of opioids for the treatment of severe pain. Glucocorticoid receptors (GRs) are among molecular substrates involved in these processes. Most studies focus on the role of neuronal GR, while the involvement of GR on glial cells is not fully understood. To address this issue, we used a transgenic model of conditional GR knockout mice, targeted to connexin 30-expressing astrocytes, treated with repeated doses of morphine. We observed no difference between control mice and astrocytic GR knockouts in the development of antinociceptive tolerance. Nevertheless, when animals were subjected to precipitated withdrawal, knockouts presented some attenuated symptoms, including jumping. Taken together, our data suggest that hippocampal and spinal astrocytic GRs appear to be involved in opioid withdrawal, and drugs targeting the GR may relieve some symptoms of morphine withdrawal without influencing its antinociceptive properties.

Molecular Modeling of µ Opioid Receptor Ligands with Various Functional Properties: PZM21, SR-17018, Morphine, and Fentanyl-Simulated Interaction Patterns Confronted with Experimental Data.

Molecular modeling approaches are an indispensable part of the drug design process. They not only support the process of searching for new ligands of a given receptor, but they also play an important role in explaining particular activity pathways of a compound. In this study, a comprehensive molecular modeling protocol was developed to explain the observed activity profiles of selected µ opioid receptor agents: two G protein-biased µ opioid receptor agonists(PZM21 and SR-17018), unbiased morphine, and the ß-arrestin-2-biased agonist,fentanyl. The study involved docking and molecular dynamics simulations carried out for three crystal structures of the target at a microsecond scale, followed by the statistical analysis of ligand-protein contacts. The interaction frequency between the modeled compounds and the subsequent residues of a protein during the simulation was also correlated with the output of in vitro and in vivo tests, resulting in the set of amino acids with the highest Pearson correlation coefficient values. Such indicated positions may serve as a guide for designing new G protein-biased ligands of the µ opioid receptor.

miR-483-5p offsets functional and behavioural effects of stress in male mice through synapse-targeted repression of Pgap2 in the basolateral amygdala.

Severe psychological trauma triggers genetic, biochemical and morphological changes in amygdala neurons, which underpin the development of stress-induced behavioural abnormalities, such as high levels of anxiety. miRNAs are small, non-coding RNA fragments that orchestrate complex neuronal responses by simultaneous transcriptional/translational repression of multiple target genes. Here we show that miR-483-5p in the amygdala of male mice counterbalances the structural, functional and behavioural consequences of stress to promote a reduction in anxiety-like behaviour. Upon stress, miR-483-5p is upregulated in the synaptic compartment of amygdala neurons and directly represses three stress-associated genes: Pgap2, Gpx3 and Macf1. Upregulation of miR-483-5p leads to selective contraction of distal parts of the dendritic arbour and conversion of immature filopodia into mature, mushroom-like dendritic spines. Consistent with its role in reducing the stress response, upregulation of miR-483-5p in the basolateral amygdala produces a reduction in anxiety-like behaviour. Stress-induced neuromorphological and behavioural effects of miR-483-5p can be recapitulated by shRNA mediated suppression of Pgap2 and prevented by simultaneous overexpression of miR-483-5p-resistant Pgap2. Our results demonstrate that miR-483-5p is sufficient to confer a reduction in anxiety-like behaviour and point to miR-483-5p-mediated repression of Pgap2 as a critical cellular event offsetting the functional and behavioural consequences of psychological stress.

Influence of G protein-biased agonists of µ-opioid receptor on addiction-related behaviors.

Opioid analgesics remain a gold standard for the treatment of moderate to severe pain. However, their clinical utility is seriously limited by a range of adverse effects. Among them, their high-addictive potential appears as very important, especially in the context of the opioid epidemic. Therefore, the development of safer opioid analgesics with low abuse potential appears as a challenging problem for opioid research. Among the last few decades, different approaches to the discovery of novel opioid drugs have been assessed. One of the most promising is the development of G protein-biased opioid agonists, which can activate only selected intracellular signaling pathways. To date, discoveries of several biased agonists acting via µ-opioid receptor were reported. According to the experimental data, such ligands may be devoid of at least some of the opioid side effects, such as respiratory depression or constipation. Nevertheless, most data regarding the addictive properties of biased µ-opioid receptor agonists are inconsistent. A global problem connected with opioid abuse also requires the search for effective pharmacotherapy for opioid addiction, which is another potential application of biased compounds. This review discusses the state-of-the-art on addictive properties of G protein-biased µ-opioid receptor agonists as well as we analyze whether these compounds can diminish any symptoms of opioid addiction. Finally, we provide a critical view on recent data connected with biased signaling and its implications to in vivo manifestations of addiction.

Knockdown of the astrocytic glucocorticoid receptor in the central nucleus of the amygdala diminishes conditioned fear expression and anxiety.

The amygdala is a key structure involved in both physiological and behavioural effects of fearful and stressful stimuli. The central stress response is controlled by the activity of the hypothalamic-pituitary-adrenal (HPA) axis via glucocorticoid hormones, acting mainly through glucocorticoid receptors (GR), widely expressed among different brain regions, including the central nucleus of the amygdala (CeA). Although to date, neuronal GR was postulated to be involved in the mediating stress effects, increasing evidence points to the vital role of glial GR. Here, we aimed to evaluate the role of astrocytic GR in CeA in various aspects of the stress response. We used a lentiviral vector to disrupt an astrocytic GR in the CeA of Aldh1l1-Cre transgenic mice. Astrocytic GR knockdown mice (GR KD) exhibited an attenuated expression of fear-related memory in the fear conditioning paradigm. Interestingly, the consolidation of non-stressful memory in the novel object recognition test remained unchanged. Moreover, GR KD group presented reduced anxiety, measured in the open field test. However, knockdown of astrocytic GR in the CeA did not affect an acute response to stress in the tail suspension test. Taken together, obtained results suggest that astrocytic GR in the CeA promotes aversive memory consolidation and some aspects of anxiety behaviour.

Comparison of an Addictive Potential of µ-Opioid Receptor Agonists with G Protein Bias: Behavioral and Molecular Modeling Studies.

Among different approaches to the search for novel-safer and less addictive-opioid analgesics, biased agonism has received the most attention in recent years. Some µ-opioid receptor agonists with G protein bias, including SR compounds, were proposed to induce diminished side effects. However, in many aspects, behavioral effects of those compounds, as well as the mechanisms underlying differences in their action, remain unexplored. Here, we aimed to evaluate the effects of SR-14968 and SR-17018, highly G protein-biased opioid agonists, on antinociception, motor activity and addiction-like behaviors in C57BL/6J mice. The obtained results showed that the compounds induce strong and dose-dependent antinociception. SR-14968 causes high, and SR-17018 much lower, locomotor activity. Both agonists develop reward-associated behavior and physical dependence. The compounds also cause antinociceptive tolerance, however, developing more slowly when compared to morphine. Interestingly, SR compounds, in particular SR-17018, slow down the development of antinociceptive tolerance to morphine and inhibit some symptoms of morphine withdrawal. Therefore, our results indicate that SR agonists possess rewarding and addictive properties, but can positively modulate some symptoms of morphine dependence. Next, we have compared behavioral effects of SR-compounds and PZM21 and searched for a relationship to the substantial differences in molecular interactions that these compounds form with the µ-opioid receptor.

Astrocytes determine conditioned response to morphine via glucocorticoid receptor-dependent regulation of lactate release.

To date, neurons have been the primary focus of research on the role of glucocorticoids in the regulation of brain function and pathological behaviors, such as addiction. Astrocytes, which are also glucocorticoid-responsive, have been recently implicated in the development of drug abuse, albeit through as yet undefined mechanisms. Here, using a spectrum of tools (whole-transcriptome profiling, viral-mediated RNA interference in vitro and in vivo, behavioral pharmacology and electrophysiology), we demonstrate that astrocytes in the nucleus accumbens (NAc) are an important locus of glucocorticoid receptor (GR)-dependent transcriptional changes that regulate rewarding effects of morphine. Specifically, we show that targeted knockdown of the GR in the NAc astrocytes enhanced conditioned responses to morphine, with a concomitant inhibition of morphine-induced neuronal excitability and plasticity. Interestingly, GR knockdown did not influence sensitivity to cocaine. Further analyses revealed GR-dependent regulation of astroglial metabolism. Notably, GR knockdown inhibited induced by glucocorticoids lactate release in astrocytes. Finally, lactate administration outbalanced conditioned responses to morphine in astroglial GR knockdown mice. These findings demonstrate a role of GR-dependent regulation of astrocytic metabolism in the NAc and a key role of GR-expressing astrocytes in opioid reward processing.

Functional characterization of a novel opioid, PZM21, and its effects on the behavioural responses to morphine.

BACKGROUND AND PURPOSE: The concept of opioid ligands biased towards the G protein pathway with minimal recruitment of ß-arrestin-2 is a promising approach for the development of novel, efficient, and potentially nonaddictive opioid therapeutics. A recently discovered biased µ-opioid receptor agonist, PZM21, showed analgesic effects with reduced side effects. Here, we aimed to further investigate the behavioural and biochemical properties of PZM21. EXPERIMENT APPROACH: We evaluated antinociceptive effects of systemic and intrathecal PZM21 administration. Its addiction-like properties were determined using several behavioural approaches: conditioned place preference, locomotor sensitization, precipitated withdrawal, and self-administration. Also, effects of PZM21 on morphine-induced antinociception, tolerance, and reward were assessed. Effects of PZM21 on striatal release of monoamines were evaluated using brain microdialysis. KEY RESULTS: PZM21 caused long-lasting dose-dependent antinociception. It did not induce reward- and reinforcement-related behaviour; however, its repeated administration led to antinociceptive tolerance and naloxone-precipitated withdrawal symptoms. Pretreatment with PZM21 enhanced morphine-induced antinociception and attenuated the expression of morphine reward. In comparison to morphine, PZM21 administration induced a moderate release of dopamine and a robust release of 5-HT in the striatum. CONCLUSIONS AND IMPLICATIONS: PZM21 exhibited antinociceptive efficacy, without rewarding or reinforcing properties. However, its clinical application may be restricted, as it induces tolerance and withdrawal symptoms. Notably, its ability to diminish morphine reward implies that PZM21 may be useful in treatment of opioid use disorders.

Glucocorticoid receptor signaling in astrocytes is required for aversive memory formation.

Stress elicits the release of glucocorticoids (GCs) that regulate energy metabolism and play a role in emotional memory. Astrocytes express glucocorticoid receptors (GR), but their contribution to cognitive effects of GC's action in the brain is unknown. To address this question, we studied how astrocyte-specific elimination of GR affects animal behavior known to be regulated by stress. Mice with astrocyte-specific ablation of GR presented impaired aversive memory expression in two different paradigms of Pavlovian learning: contextual fear conditioning and conditioned place aversion. These mice also displayed compromised regulation of genes encoding key elements of the glucose metabolism pathway upon GR stimulation. In particular, we identified that the glial, but not the neuronal isoform of a crucial stress-response molecule, Sgk1, undergoes GR-dependent regulation in vivo and demonstrated the involvement of SGK1 in regulation of glucose uptake in astrocytes. Together, our results reveal astrocytes as a central element in GC-dependent formation of aversive memory and suggest their relevance for stress-induced alteration of brain glucose metabolism. Consequently, astrocytes should be considered as a cellular target of therapies of stress-induced brain diseases.