Roles of lncLingo2 and its derived miR-876-5p in the acquisition of opioid reinforcement.

Recent studies found that non-coding RNAs (ncRNAs) played crucial roles in drug addiction through epigenetic regulation of gene expression and underlying drug-induced neuroadaptations. In this study, we characterized lncRNA transcriptome profiles in the nucleus accumbens (NAc) of mice exhibiting morphine-conditioned place preference (CPP) and explored the prospective roles of novel differentially expressed lncRNA, lncLingo2 and its derived miR-876-5p in the acquisition of opioids-associated behaviours. We found that the lncLingo2 was downregulated within the NAc core (NAcC) but not in the NAc shell (NAcS). This downregulation was found to be associated with the development of morphine CPP and heroin intravenous self-administration (IVSA). As Mfold software revealed that the secondary structures of lncLingo2 contained the sequence of pre-miR-876, transfection of LV-lncLingo2 into HEK293 cells significantly upregulated miR-876 expression and the changes of mature miR-876 are positively correlated with lncLingo2 expression in NAcC of morphine CPP trained mice. Delivering miR-876-5p mimics into NAcC also inhibited the acquisition of morphine CPP. Furthermore, bioinformatics analysis and dual-luciferase assay confirmed that miR-876-5p binds to its target gene, Kcnn3, selectively and regulates morphine CPP training-induced alteration of Kcnn3 expression. Lastly, the electrophysiological analysis indicated that the currents of small conductance calcium-activated potassium (SK) channel was increased, which led to low neuronal excitability in NAcC after CPP training, and these changes were reversed by lncLingo2 overexpression. Collectively, lncLingo2 may function as a precursor of miR-876-5p in NAcC, hence modulating the development of opioid-associated behaviours in mice, which may serve as an underlying biomarker and therapeutic target of opioid addiction.

The Role of circTmeff-1 in Morphine Addiction Memory of Mice.

In addition to the essential pharmacological effects of opioids, situational cues associated with drug addiction memory are key triggers for drug seeking. CircRNAs, an emerging hotspot regulator in crown genetics, play an important role in central nervous system-related diseases. However, the internal mediating mechanism of circRNAs in the field of drug reward and addiction memory remains unknown. Here, we trained mice on a conditional place preference (CPP) model and collected nucleus accumbens (NAc) tissues from day 1 (T0) and day 8 (T1) for high-throughput RNA sequencing. QRT-PCR analysis revealed that circTmeff-1 was highly expressed in the NAc core but not in the NAc shell, suggesting that it plays a role in addiction memory formation. Meanwhile, the down-regulation of circTmeff-1 by adeno-associated viruses in the NAc core or shell could inhibit the morphine CPP scores. Subsequently, the GO and KEGG analyses indicated that circTmeff-1 might regulate the addiction memory via the MAPK and AMPK pathways. These findings suggest that circTmeff-1 in NAc plays a crucial role in morphine-dependent memory formation.

CircTmeff-1 in the nucleus accumbens regulates the reconsolidation of cocaine-associated memory.

Reconsolidation of drug memories is the process of restoring unstable memories after unconditioned (UCS; e.g., drugs) or conditioned stimulus (CS; e.g., drug-paired contexts), and provides promise for prevention of drug relapse. Circular RNAs (circRNAs) have important effects on the transcription and post-transcriptional regulation of gene expression. However, the role of circRNAs in the reconsolidation of drug memories is unclear. Here, we observed that cocaine-induced memory retrieval significantly increased circTmeff-1 level in the nucleus accumbens (NAc) core but not shell. Importantly, the disrupted expression of circTmeff-1 using virus in the NAc core damaged the reconsolidation of cocaine-associated memories. The knockdown of circTmeff-1 in the NAc shell or without UCS retrieval or 9 h after UCS retrieval had no such effects. Mechanistically, using bioinformatic analysis and loss- or gain- of function assays, we revealed that antagomiR-206 reversed the inhibitory effect of circTmeff-1 knockdown on the expression of brain-derived neurotrophic factor (BDNF) during the reconsolidation of cocaine-associated memories. Taken together, these results demonstrate the role of circTmeff-1 in the reconsolidation of cocaine-associated memory and that circTmeff-1 may function as a decoy for miR-206 to regulate the expression of BDNF.

Roles of miR-592-3p and Its Target Gene, TMEFF1, in the Nucleus Accumbens During Incubation of Morphine Craving.

BACKGROUND: Prolonged forced abstinence from morphine can increase cue-induced cravings for the drug, contributing to a persistent vulnerability to relapse. Previous studies have identified the implications of aberrant microRNA (miRNA) regulation in the pathogenesis of morphine addiction, but the changes in miRNA expression during the incubation of morphine craving are still unknown. METHODS: Nucleus accumbens (NAc)-specific altered miRNA transcriptomics was determined in a mouse model of cue-induced incubation of morphine craving following a next-generation sequencing method and verified by RT-qPCR. Bioinformatics analysis was performed to predict the target gene of selected miRNA, and the protein expression of the target gene was detected by western blot. A dual-luciferase assay was performed to confirm the binding sites, and gain- and loss-of-function strategy was applied to understand the mechanism of miRNA and its target gene. RESULTS: The miR-592-3p observed to be downregulated in the NAc core was linked to the incubation of morphine craving, and a dual-luciferase assay was performed to confirm the binding sites of miR-592-3p in its target gene, tomoregulin-1 (TMEFF1). Also, gain- and loss-of-function analyses revealed that the inhibition of miR-592-3p expression in the NAc core negatively regulated TMEFF1 expression, thereby enhancing the incubation of morphine craving; however, the overexpression of miR-592-3p in the NAc core resulted in a decreased expression of TMEFF1, thereby reducing the incubation of morphine craving. CONCLUSION: Our findings demonstrated that miR-592-3p can improve the incubation of morphine craving by targeting TMEFF1, and thus, it holds a therapeutic potential to inhibit opioid craving.

The role of circTmeff-1 in incubation of context-induced morphine craving.

A progressive increase in drug craving following drug exposure is an important trigger of relapse. CircularRNAs (CircRNAs), key regulators of gene expression, play an important role in neurological diseases. However, the role of circRNAs in drug craving is unclear. In the present study, we trained mice to morphine conditioned place preference (CPP) and collected the nucleus accumbens (NAc) sections on abstinence day 1 (AD1) and day 14 (AD14) for RNA-sequencing. CircTmeff-1, which was highly expressed in the NAc core, was associated with incubation of context-induced morphine craving. The gain- and loss- of function showed that circTmeff-1 was a positive regulator of incubation. Simultaneously, the expression of miR-541-5p and miR-6934-3p were down-regulated in the NAc core during the incubation period. The dual luciferase reporter, RNA pulldown, and fluorescence insitu hybridization assays confirmed that miR-541-5p and miR-6934-3p bind to circTmeff-1 selectively. Furthermore, bioinformatics and western blot analysis suggested that vesicle-associated membrane protein 1 (VAMP1) and neurofascin (NFASC), both overlapping targets of miR-541-5p and miR-6934-3p, were highly expressed during incubation. Lastly, AAV-induced down-regulation of circTmeff-1 decreased VAMP1 and NFASC expression and incubation of morphine craving. These findings suggested that circTmeff-1, a novel circRNA, promotes incubation of context-induced morphine craving by sponging miR-541/miR-6934 in the NAc core. Thus, circTmeff-1 represents a potential therapeutic target for context-induced opioid craving, following prolonged abstinence.

Identification and Characterization of Biomarkers and Their Role in Opioid Addiction by Integrated Bioinformatics Analysis.

Although numerous studies have confirmed that the mechanisms of opiate addiction include genetic and epigenetic aspects, the results of such studies are inconsistent. Here, we downloaded gene expression profiling information, GSE87823, from the Gene Expression Omnibus database. Samples from males between ages 19 and 35 were selected for analysis of differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses were used to analyze the pathways associated with the DEGs. We further constructed protein-protein interaction (PPI) networks using the STRING database and used 10 different calculation methods to validate the hub genes. Finally, we utilized the Basic Local Alignment Search Tool (BLAST) to identify the DEG with the highest sequence similarity in mouse and detected the change in expression of the hub genes in this animal model using RT-qPCR. We identified three key genes, ADCY9, PECAM1, and IL4. ADCY9 expression decreased in the nucleus accumbens of opioid-addicted mice compared with control mice, which was consistent with the change seen in humans. The importance and originality of this study are provided by two aspects. Firstly, we used a variety of calculation methods to obtain hub genes; secondly, we exploited homology analysis to solve the difficult challenge that addiction-related experiments cannot be carried out in patients or healthy individuals. In short, this study not only explores potential biomarkers and therapeutic targets of opioid addiction but also provides new ideas for subsequent research on opioid addiction.

Methamphetamine induces GSDME-dependent cell death in hippocampal neuronal cells through the endoplasmic reticulum stress pathway.

Methamphetamine (METH) is an illegal amphetamine-typed psychostimulant that is abused worldwide and causes serious public health problems. METH exposure induces apoptosis and autophagy in neuronal cells. However, the role of pyroptosis in METH-induced neurotoxicity is still unclear. Here, we investigate whether pyroptosis is involved in METH-induced hippocampal neurotoxicity and the potential mechanisms of Endoplasmic reticulum (ER) stress in hippocampal neuronal cells. For this purpose, the expression levels of pyroptosis-related proteins, GSDMD and GSDME, were analyzed by immunoblotting and immunohistochemistry in the hippocampal neuron cell line HT-22. Next, we explored METH-induced pyroptosis in HT-22 using immunoblotting, LDH assays and SYTOX green acid staining. Further, the relationship between pyroptosis and ER stress in METH-induced hippocampal neuron damage was studied in HT-22 cells using inhibitors including TUDCA, a specific inhibitor of ER stress, GSK-2656157, a PERK pathway inhibitor and STF-0803010, an inhibitor of IRE1α endoribonuclease activity. This relationship was also studied using siRNAs, including siTRAF2, an siRNA against IRE1α kinase activity and siATF6 against the ATF6 pathway, which were analyzed by immunoblotting, LDH assays and SYTOX green acid staining. GSDME but not GSDMD was found to be expressed in HT-22 cells. METH treatment induced the upregulation of cleaved GSDME-NT and LDH release, as well as the increase of SYTOX green positive cells in HT-22 cells, which was partly reversed by inhibitors and siRNAs, indicating that the ER stress signaling pathway was involved in GSDME-dependent cell death induced by METH. In summary, these results revealed that METH induced ER stress that mediated GSDME-dependent cell death in hippocampal neuronal cells. These findings provide novel insight into the mechanisms of METH-induced neurotoxicity.

Berberine Facilitates Extinction of Drug-Associated Behavior and Inhibits Reinstatement of Drug Seeking.

A high rate of relapse is a major clinical problem among drug-addicted individuals. Persistent traces of drug-associated reward memories contribute to intense craving and often trigger relapse. A number of interventions on drug-associated memories have shown significant benefits in relapse prevention. Among them are pre- or post-extinction pharmacological manipulations that facilitate the extinction of drug-associated behavior. Berberine, a bioactive isoquinoline alkaloid, has been recently reported to provide therapeutic benefits for a number of central nervous system (CNS) disorders, including morphine addiction. The present study aimed to investigate whether berberine could serve as a post-extinction pharmacological intervention agent to reduce risks of reinstatement of drug seeking. We found that an intragastric administration of berberine at doses of 25 and 50 mg/kg during the critical time window significantly facilitated the extinction of morphine-reward related behavior in free access and confined conditioned place preference (CPP) extinction paradigms, and subsequently, it prevented reinstatement and spontaneous recovery of morphine-induced CPP in mice. Intriguingly, the berberine treatment with or without extinction training altered expression of plasticity-related proteins such as brain-derived neurotrophic factor (BDNF), AMPA receptors (GluA1 and GluA2) in the nucleus accumbens (NAc). Moreover, the post-extinction berberine treatment significantly reduced reinstatement of cocaine-induced CPP and operant intravenous self-administration (IVSA) memories in rats. Altogether, our findings suggest that extinction training combined with the post-extinction berberine treatment can facilitate extinction of drug-associated behavior making it an attractive therapeutic candidate in relapse prevention.

Effects of exogenous cholecystokinin octapeptide on acquisition of naloxone precipitated withdrawal induced conditioned place aversion in rats.

Cholecystokinin octapeptide (CCK-8), a gut-brain peptide, regulates a variety of physiological behavioral processes. Previously, we reported that exogenous CCK-8 attenuated morphine-induced conditioned place preference, but the possible effects of CCK-8 on aversively motivated drug seeking remained unclear. To investigate the effects of endogenous and exogenous CCK on negative components of morphine withdrawal, we evaluated the effects of CCK receptor antagonists and CCK-8 on the naloxone-precipitated withdrawal-induced conditioned place aversion (CPA). The results showed that CCK2 receptor antagonist (LY-288,513, 10 µg, i.c.v.), but not CCK1 receptor antagonist (L-364,718, 10 µg, i.c.v.), inhibited the acquisition of CPA when given prior to naloxone (0.3 mg/kg) administration in morphine-dependent rats. Similarly, CCK-8 (0.1-1 µg, i.c.v.) significantly attenuated naloxone-precipitated withdrawal-induced CPA, and this inhibitory function was blocked by co-injection with L-364,718. Microinjection of L-364,718, LY-288,513 or CCK-8 to saline pretreated rats produced neither a conditioned preference nor aversion, and the induction of CPA by CCK-8 itself after morphine pretreatments was not significant. Our study identifies a different role of CCK1 and CCK2 receptors in negative affective components of morphine abstinence and an inhibitory effect of exogenous CCK-8 on naloxone-precipitated withdrawal-induced CPA via CCK1 receptor.

The effects of exogenous CCK-8 on the acquisition and expression of morphine-induced CPP.

Cholecystokinin octapeptide (CCK-8) is the most potent endogenous anti-opioid peptide and regulates a variety of physiological processes. In our previous study, we found that exogenous CCK-8 attenuated naloxone-induced withdrawal symptoms, but the possible regulative effects of CCK-8 on the rewarding effects of morphine were not examined. In the present study, we aimed to determine the exact effects of exogenous CCK-8 at various doses on the rewarding action of morphine by utilizing the unbiased conditioned place preference (CPP) paradigm. We therefore examined the effects of CCK-8 on the acquisition, expression and extinction of morphine-induced CPP and on locomotor activity. The results showed that CCK-8 (0.01-1µg, i.c.v.), administered alone, induced neither CPP nor place aversion, but blocked the acquisition of CPP when administered with 10mg/kg morphine. The highest dose of CCK-8 (1µg) administered before CPP testing increased CPP and, along with lower doses (0.1µg), reduced its extinction. In addition, the highest dose (1µg) of CCK-8 suppressed locomotor activity. Our study provides the first behavioral evidence for the inhibitory effects of exogenous CCK-8 on rewarding activity and reveals significant effects of exogenous CCK-8 on various stages of place preference and the development of opioid dependence.