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Background Mitochondrial dynamics in neurons accompanied by neuroinflammation has been proved as pivotal events during repeated morphine exposure, however, the relationship between mitochondrial dynamics and neuroinflammation still remains unknown. Methods This study was designed to investigate the potential role of astrocyte Drp1 in neuroinflammation during morphine addiction. Nucleus accumbens (NAc) tissues were collected for immunofluorescence, transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qRT-PCR) to detect the expression of pro-inflammatory cytokines and mitochondrial fission proteins. Morphine-induced conditioned place preference (CPP) and open field test (OFT) were used to determine the effects of Mdivi-1, a selective inhibitor of mitochondrial fission protein Drp1 in the rewarding properties of morphine. Astrocyte-specific knockdown experiments by an adeno-associated virus (AAV) vector containing shRNADrp1–EGFP infusion were performed to determine the effects of astrocyte Drp1 in NAc of mice with morphine treatment. Results In this study, we found that repeated morphine exposure induced mitochondrial fragmentation in neurons, astrocytes, and microglia in NAc, correlating with increased inflammatory markers and addictive behaviors. The application of Mdivi-1 effectively mitigated mitochondrial fragmentation and astrocyte-mediated neuroinflammation within the NAc, thereby alleviating morphine-induced addictive behaviors. Crucially, the astrocyte-specific knockdown of Drp1 in NAc significantly curtailed drug-seeking behavior and substantially reduced neuroinflammation. Conclusions Collectively, our findings suggest that alterations in mitochondrial dynamics, particularly within astrocytes, play an important role in regulating neuroinflammation associated with morphine addiction. This research offers novel insights into potential therapeutic strategies for addressing substance use disorder (SUD) by regulating mitochondrial dynamics within astrocyte.

Background Physical symptoms and aversion induced by opioid withdrawal strongly affect the management of opioid addiction. YTH N6-methyladenosine (m 6 A) RNA binding protein 1 (YTHDF1), an m 6 A-binding protein, from the periaqueductal gray (PAG) reportedly contributes to morphine tolerance and hyperalgesia. However, the role of YTHDF1 in morphine withdrawal remains unclear. Methods A naloxone-precipitated morphine withdrawal model was established in C57/BL6 mice or transgenic mice. YTHDF1 was knocked down via adeno-associated virus transfection. Combined with the results of the single-cell RNA sequencing analysis, the changes in morphine withdrawal somatic signs and conditioned place aversion (CPA) scores were compared when YTHDF1 originating from different neurons in the ventrolateral periaqueductal gray (vlPAG) was knocked down. We further explored the role of inflammatory factors and transcription factors related to inflammatory response in morphine withdrawal. Results Our results revealed that YTHDF1 expression was upregulated in the vlPAG of mice with morphine withdrawal and that the knockdown of vlPAG YTHDF1 attenuated morphine withdrawal-related somatic signs and aversion. The levels of NF-κB and p-NF-κB were reduced after the inhibition of YTHDF1 in the vlPAG. YTHDF1 from vlPAG inhibitory neurons, rather than excitatory neurons, facilitated morphine withdrawal responses. The inhibition of YTHDF1 in vlPAG somatostatin (Sst)-expressing neurons relieved somatic signs of morphine withdrawal and aversion, whereas the knockdown of YTHDF1 in cholecystokinin (Cck)-expressing or parvalbumin (PV)-expressing neurons did not change morphine withdrawal-induced responses. The activity of c-fos + neurons, the intensity of the calcium signal, the density of dendritic spines, and the frequency of mIPSCs in the vlPAG, which were increased in mice with morphine withdrawal, were decreased with the inhibition of YTHDF1 from vlPAG inhibitory neurons or Sst-expressing neurons. Knockdown of NF-κB in Sst-expressing neurons also alleviated morphine withdrawal-induced responses. Conclusions YTHDF1 originating from Sst-expressing neurons in the vlPAG is crucial for the modulation of morphine withdrawal responses, and the underlying mechanism might be related to the regulation of the expression and phosphorylation of NF-κB.

The accumulated evidence suggests that varying levels of tyrosine kinase receptor signaling pathway activity may regulate opiate-associated neuroadaptation of noradrenergic system. Neurotrophin-3 (NT-3) interacts with tropomyosin receptor kinases (TRKs), binding mainly to TRKC receptors, which are expressed within noradrenergic neurons in the blue spot (locus coeruleus, LC). Considering the difficulties in delivering full-length neurotrophins to the CNS after systemic administration, low-molecular mimetics of loop 4 in NT-3, hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301), and hexamethylenediamide bis-(N-γ-oxybutyryl-L-glutamyl-L-asparagine) (GTS-302), activating TRKC and TRKB receptors, were synthesized. The aim of the study is comparative examination of the effects of NT-3 dipeptide mimetics on the signs of morphine withdrawal in outbred white rats with opiate dependence, as well as investigation of activation of postreceptor signaling pathways by the mimetics. Dipeptides GTS-301 and GTS-302 after acute administration at doses of 0.1, 1.0, and 10.0 mg/kg (i.p., intraperitoneal) had a dose-dependent effect on the specific morphine withdrawal symptoms with the most effective dose being 1.0 mg/kg. Maximum decrease in the total index of morphine withdrawal syndrome for GTS-301 was 31.3% and for GTS-302 – 41.4%. Unlike GTS-301, GTS-302 weakened mechanical allodynia induced by morphine withdrawal, reducing tactile sensitivity. When studying activation of the postreceptor signaling pathways by the NT-3 mimetics in the HT-22 hippocampal cell culture, a different pattern of postreceptor signaling was shown: GTS-302 (10−6 M), similar to NT-3, activates all three MAPK/ERK, PI3K/AKT/mTOR, and PLCγ1 pathways, while GTS-301 (10−6 M) triggers only MAPK/ERK and PLCγ1 pathways. Thus, the identified features of attenuation of the morphine withdrawal syndrome in the rats under GTS-301 and GTS-302 effects could be associated with different activation pattern of the postreceptor pathways.

Background: Anxiety is a negative emotion that contributes to craving and relapse during drug withdrawal. Sirtuins 1 (SIRT1) has been reported to be critical in both negative emotions and drug addiction. However, it remains incompletely elucidated whether SIRT1 is involved in morphine withdrawal-associated anxiety. Methods: We established a mouse model of anxiety-like behaviors induced by morphine withdrawal and then detected neuronal activity with immunofluorescence and mitochondrial morphology with electron microscopy, mitochondrial DNA contents with quantitative real-time PCR, and mitochondrial function with the ATP content detection kit and the Mitochondrial Complex IV Activity Kit in the basolateral amygdala (BLA). The mitochondrial molecules were detected by western blot. Then we used virus-mediated downregulation and overexpression of SIRT1 in BLA to investigate the effect of SIRT1 on anxiety and mitochondrial function. Finally, we examined the effects of pharmacological inhibition of SIRT1 on anxiety and mitochondrial function. Results: We found that BLA neuronal activity, mitochondrial function, and mtDNA content were significantly higher in morphine withdrawal mice. Furthermore, the expression levels of mitochondrial molecules increased in BLA cells. Virus-mediated downregulation of SIRT1 in BLA prevented anxiety-like behaviors in morphine withdrawal mice, whereas overexpression of SIRT1 in BLA facilitated anxiety-like behaviors in untreated mice through the SIRT1/ peroxisome proliferator activated receptor gamma coactivator 1-alpha pathway. Intra-BLA infusion of selective SIRT1 antagonist EX527 effectively ameliorated anxiety-like behaviors and mitochondrial dysfunction in mice with morphine withdrawal. Conclusion: Our results implicate a causal role for SIRT1 in the regulation of anxiety through actions on mitochondrial biogenesis. Inhibitors targeting SIRT1 may have therapeutic potential for the treatment of opioid withdrawal-associated anxiety. Keywords: Anxiety, morphine withdrawal, basolateral amygdala, mitochondria, SIRT1

Abstract 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.

ɣ-aminobutyric acid (GABA) facilitator valproic acid may be able to curb memory disruption induced by morphine exposure. The effects of the GABA facilitator valproic acid on the behavioral tolerance induced by morphine were investigated. Then hippocampal-dependent tasks named spatial-working and short-term memory procedures using the Y-maze apparatus were examined in morphine tolerant rats. Finally, the changes in the expression of hippocampal GABA-A receptors underlying morphine tolerance were also examined. Rats were treated with daily morphine injections, with or without distinct contextual pairing. To examine the effect of valproic acid on morphine tolerance expression, valproic acid was pretreated an hour before morphine. Spatial-working and short-term memory procedures using the Y-maze apparatus were examined in morphine tolerant rats. Afterwards the changes in the expression of hippocampal GABAα receptors using the quantitative real-time PCR and western blot techniques to detect GABArα subunits mRNAs and protein level were studied. Our results showed that both learned and non-associative morphine tolerance influence short-term memory and the subjacent expression of GABArα mRNAs and protein level. Despite its attenuating effects on the development and expression of both learned and non-associative morphine tolerance, only associative morphine tolerance-induced memory dysfunction was ameliorated by valproic acid pretreatment. We also found that the expression of GABArα1, α2, α5 subunits mRNAs and GABAα protein level were affected heavier in associative morphine tolerant rats. Our data supports the hypothesis that unconditioned and learned morphine tolerance influences short-term memory and the expression of GABArα 1, α2, α5 mRNAs and GABArα protein level differently, and adds to our understanding of the behavioral and molecular aspects of the learned tolerance to morphine effects.

Topiramate evokes pharmacological activity via a blockade of voltage-dependent sodium channels, reduction in glutamate release, inhibition of AMPA receptors and kainate receptors, and potentiation of GABAergic neurotransmission. Therefore, it is used not only as an antiseizure drug but is also effective in migraine prophylaxis, cluster headaches, neuropathic pain, and alcohol dependence. The aim of this study was to investigate the effect of topiramate in morphine dependence in mice, particularly in terms of morphine tolerance, morphine withdrawal signs, and morphine sensitization. In these experiments, topiramate was administered both acutely and chronically. Topiramate significantly reduced the morphine tolerance in the hot-plate test and attenuated naloxone-induced morphine withdrawal signs. Its effect on morphine sensitization to the locomotor activity of mice was poor. The obtained results showed that topiramate might be an effective drug for reducing the physical symptoms of morphine dependence.

Background/Objectives: Situations previously paired with drug use can become conditioned stimuli (i.e., physical stress or psychosocial stress) that elicit intense craving and relapse, even after prolonged abstinence. Previous studies have shown that pharmacological disruption of reconsolidation after memory reactivation could be promising for reducing pathological fear and stress-related responses. For this reason, the aim of this research was to examine the role of β-AR in the retrieval of aversive memories through the potential of β-AR antagonism to mitigate the effects of exposure to stressful stimuli. Methods: This question was addressed using a model to assess the re-emergence of an aversive contextual memory induced by both physical stressors (restraint and tail-pinch) and psychosocial stress (social defeat) in morphine- or saline-treated mice previously subjected to a conditioned place aversion (CPA) paradigm, in which naloxone was administered to precipitate opioid withdrawal. To assess the effects of propranolol on aversive memories related to opioid addiction, the number of chamber crossings and the time spent in the naloxone-paired compartment were measured. Results: Our results showed that morphine-treated mice spent significantly less time in the naloxone-paired chamber than saline mice during the post-test and after exposure to stressful stimuli, than during the pre-test, showing an effect for aversive memories in addiction. In contrast, when propranolol was administered intraperitoneally 30 min before the exposure to both social and physical stress, the time spent enhanced significantly (p < 0.01), supporting a role for propranolol in addiction-related memories. Conclusions: These results suggest that propranolol could attenuate the aversive memories that may contribute to relapse to opioid addiction.

Objective: The effects of morphine addiction on testicular activity are significant. This study aimed to examine testicular contraction and tissue changes that may occur in the testicles of rats with experimentally induced morphine addiction. Method: Thirty-two Wistar albino rats were divided into four groups: Control (C), Morphine Dependence (M), Morphine Dependence + Morphine Withdrawal (MN), and Morphine Withdrawal (N). Morphine dependence was induced in the M and MN groups. Before decapitation, the rats were evaluated for behavioral changes and scored according to the Geller-Holtzman scale. After decapitation, one testicular tissue sample was placed in Krebs solution for contraction analysis, while the other was fixed in formaldehyde for histopathological examination. Testicular capsule contraction tensions, withdrawal scores, and modified Johnsen scores were assessed. Results: When the effects of morphine withdrawal were examined in rats injected with morphine, a significant difference was found between the C and MN, C and N, and M and MN groups (p<0.001). In isolated organ bath experiments, a significant difference in contraction values before drug application was observed between the C and MN groups (p<0.05); however, no significant difference was found in group-time values (p>0.05). Histological analyses of testicular tissue sections from the morphine group revealed a decrease in germinal layer thickness and degeneration in the seminiferous tubules. Histopathological examination of testicular tissues showed that the M group exhibited negative effects, while these effects were reversed in the MN group. Conclusion: At the cellular level, morphine was observed to have negative effects on testicular function, which could be reversed with naloxone. The testicular capsule contraction parameter was not clearly informative. In the morphine addiction model, more detailed insights into testicular contraction functions may be obtained by extending the application period and conducting dose-dependent studies. Keywords: Isolated organ bath, morphine dependence, morphine withdrawal, naloxone, opioid, testicular contraction

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.