Chronic stress promotes alcohol use and relapse, yet the mechanisms linking stress-induced anxiety to excessive ethanol consumption remain poorly understood. Plasminogen activator inhibitor-1 (PAI-1), a stress-responsive regulator of neuroplasticity, has recently emerged as a potential therapeutic target; however, its role in stress-induced alcohol-related behaviors remains unknown. This study investigated whether TM5441, a selective inhibitor of plasminogen activator inhibitor-1 (PAI-1), modulates anxiety-like behavior and ethanol consumption following chronic social stress. Male mice were subjected to social defeat chronic stress (SDCS) and treated with TM5441. Behavioral assessments included the elevated plus maze (EPM), open field test (OFT), and two-bottle choice paradigms to assess voluntary intake of ethanol, saccharin, and quinine. Blood ethanol concentration (BEC) was measured to evaluate pharmacokinetic parameters. TM5441 significantly attenuated SDCS-induced anxiety-like behaviors and restored stress-induced increases in ethanol intake and preference toward control levels. These effects were specific to ethanol, as neither TM5441 nor stress altered saccharin or quinine consumption. Importantly, pharmacokinetic analyses revealed no differences in ethanol absorption or clearance, and BEC did not correlate with ethanol intake or preference, indicating that the observed behavioral effects are unlikely to result from differences in ethanol pharmacokinetics. Correlational analyses supported a functional link between anxiety-like behavior and increased ethanol consumption under stress. Together, these findings suggest that PAI-1 may contribute to the link between stress-induced anxiety and alcohol consumption and support further investigation of PAI-1 as a potential target for stress-related alcohol use disorders.

Cocaine use disorder is a major public health issue with no approved pharmacological treatments. Drug-associated contextual memories can trigger intense craving and relapse. Although CD81 has been linked to cocaine-induced locomotion, its role in conditioned reward and relapse remains unclear. This study investigated the role of CD81 expression in the nucleus accumbens (NAc) during the acquisition and drug-primed reinstatement of cocaine-conditioned place preference (CPP) in rats. Adult male Wistar rats underwent viral manipulation of CD81 in the NAc via lentiviral vectors to induce either knockdown (shRNA) or overexpression. These manipulations were performed either before cocaine conditioning (10 mg/kg) to assess acquisition or after extinction and prior to a cocaine priming injection (5 mg/kg) to assess reinstatement. CD81 mRNA levels were measured postmortem, and Pearson correlation analyses were conducted between gene expression and behavioural outcomes. CD81 knockdown significantly reduced CPP acquisition and reinstatement, whereas CD81 overexpression increased conditioned preference in both phases. Doxycycline treatment suppressed transgene expression and abolished the behavioural effects of CD81 overexpression. Correlation analyses revealed a significant positive association between CD81 mRNA levels and CPP magnitude. These findings indicate that CD81 contributes to modulation of cocaine-conditioned reward and relapse-like behaviour within the NAc. Further investigation in additional preclinical models will be necessary to clarify the broader relevance of CD81 in addiction-related neuroplasticity.

Chronic unpredictable mild stress (CUMS) is a validated model of anxiety- and depression-like disorders and is associated with impaired neuroplasticity. Dysregulation of the plasminogen activator system, particularly elevated plasminogen activator inhibitor-1 (PAI-1), may disrupt tissue plasminogen activator (tPA)-dependent brain-derived neurotrophic factor (BDNF) signaling and contribute to stress-induced behavioral deficits. However, the therapeutic relevance of PAI-1 inhibition in mood disorders remains poorly defined. This study examined whether pharmacological inhibition of PAI-1 using TM5441 could alleviate CUMS-induced anxiety- and depression-like behaviors by modulating the hippocampal PAI-1/tPA/BDNF axis. Male and female C57BL/6 mice were exposed to CUMS for 3 weeks. TM5441, a selective SERPINE1 (PAI-1) inhibitor, was administered intraperitoneally (5 mg/kg) 20 min before behavioral testing, based on established efficacy and safety. Control mice received the vehicle (DMSO diluted in saline). Anxiety- and depression-like behaviors were assessed using the elevated plus maze, open field, sucrose preference, tail suspension, and forced swim tests. Hippocampal PAI-1, tPA, and BDNF levels were quantified, and correlations with behavioral outcomes were analyzed. TM5441 reversed CUMS-induced anxiety-like behaviors and depression-like responses. At the molecular level, TM5441 normalized stress-induced elevations in hippocampal PAI-1, restored tPA expression, and increased BDNF levels. Significant correlations were observed between PAI-1, tPA, and BDNF, as well as between hippocampal molecular markers and behavioral measures. PAI-1 inhibition by TM5441 alleviates stress-induced behavioral deficits by restoring hippocampal tPA/BDNF signaling, highlighting the PAI-1/tPA axis as a promising therapeutic target for stress-related mood disorders.

The plasminogen activator system (PAS), comprising urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their receptors, and endogenous inhibitors, has been extensively investigated for its established roles in haemostasis, fibrinolysis, vascular remodelling and extracellular matrix (ECM) homeostasis. Increasing evidence indicates that the functions of the PAS extend well beyond the cardiovascular system and have important regulatory roles in neuronal plasticity, synaptic remodelling, neuroinflammation, and neurotrophic signalling. These processes are increasingly recognized as central contributors to the neurobiological adaptations underlying substance use disorders (SUDs). In this narrative review, we critically evaluate the current evidence regarding the involvement of the PAS in SUDs, with particular emphasis on the molecular and cellular mechanisms through which uPA and tPA influence addiction-related neuroplasticity. The available literature is predominantly derived from preclinical studies, while direct clinical evidence remains limited. Experimental findings support roles for uPA and tPA in modulating reward-related circuitry, behavioural sensitization, relapse-like behaviours, and neurotrophic signalling, including potential interactions with brain-derived neurotrophic factor (BDNF)-related pathways, although the relative contributions of plasmin-dependent and plasmin-independent pathways remain incompletely understood. We also discuss the potential involvement of the PAS in neuroinflammatory responses and synaptic remodelling, together with the challenges associated with translating these findings into clinically relevant biomarkers or therapeutic strategies. Finally, we identify important gaps in current knowledge, including the need for independent replication, mechanistic clarification, and well-designed human studies to establish the clinical relevance of PAS dysregulation in addiction. Collectively, the available evidence supports a modulatory role for the PAS in addiction-related neurobiology and provides a rationale for further translational investigation, while highlighting that PAS-directed therapeutic approaches remain experimental and require substantial preclinical and clinical validation.

MicroRNAs (miRNAs) are critical regulators of gene expression and have been implicated in the pathophysiology of mood disorders and addiction. Among these, miR-124 is known to modulate synaptic plasticity and neuronal function, yet its specific role in methamphetamine (METH) reward remains poorly understood. This study aimed to investigate the role of miR-124 and its downstream target, brain-derived neurotrophic factor (BDNF), in the nucleus accumbens (NAcc) in mediating METH-induced conditioned place preference (CPP). Using a combination of behavioral assays, RT-PCR, and lentiviral-mediated gene manipulation, we examined the effects of miR-124 gain-and loss-of-function, as well as BDNF overexpression, on METH-CPP in male and female rats. METH treatment elicited robust CPP in both sexes, with no significant sex differences. RT-PCR analysis revealed that METH exposure increased miR-124 expression while decreasing BDNF mRNA levels in the NAcc, with a strong negative correlation between the two. miR-124 knockdown reduced METH-CPP, increased BDNF expression, and reversed the negative correlation, whereas miR-124 overexpression enhanced CPP, decreased BDNF, and reinforced the correlation. Importantly, BDNF overexpression in the NAcc reduced METH-CPP, mimicking the effects of miR-124 knockdown. These findings demonstrate that miR-124 enhances METH reward by suppressing BDNF expression in the NAcc, highlighting a critical miR-124/BDNF regulatory axis in addiction. This study provides novel insights into the molecular mechanisms underlying METH reward and highlights the miR-124/ BDNF axis as a pathway that may warrant further investigation in the context of substance use disorders.

This narrative review explores the intricate relationship between the plasminogen activator system (PAS), comprising urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), and a range of neuropsychiatric disorders, including depression and anxiety. By synthesizing existing preclinical and clinical evidence, we clarify the roles of uPA and tPA in the pathogenesis and potential treatments of these conditions. This narrative review emphasizes their involvement in modulating neuronal plasticity, synaptic remodeling, and neurotransmitter systems, which are pivotal in maintaining brain function and behavior. Additionally, this review highlights key mechanisms by which these activators influence the neurobiological processes underlying mood and cognitive dysfunction. Critical analysis identifies areas of consensus, such as the role of plasminogen activators in neuroinflammation and stress responses, while also addressing gaps and controversies in the literature. The findings underscore the therapeutic potential of targeting the uPA/tPA system for innovative interventions. By offering a nuanced understanding of their contributions to mood disorders, this review aims to inspire future research toward developing novel, mechanism-based treatment strategies that harness the PAS’ capacity to restore neural homeostasis and improve patient outcomes.

Understanding the molecular mechanisms underlying anxiety and ethanol-related behaviors is crucial for developing effective therapeutic interventions. This study identifies a novel role for microRNA miR-181a and its target, Sirtuin 1 (SIRT-1), in the hippocampus as contributors to anxiety-like behavior and voluntary ethanol intake. Using male and female C57BL/6 mice, we explored the causal relationship between hippocampal miR-181a expression and these behaviors. Lentivirus vectors were delivered into the hippocampus for focal miR-181a overexpression in mice. Then behaviors were observed by elevated plus maze (EPM) and open field (OF) tests. Results showed that the viral approach employed to overexpress miR-181a, in the hippocampus, resulted in increased anxiety-like behavior in the EPM and OF tests. Additionally, miR-181a overexpression exacerbated voluntary ethanol intake and preference in the two-bottle choice paradigm without affecting saccharin or quinine consumption. Mechanistically, miR-181a gain-of-function reduced SIRT-1 expression in the hippocampus. These findings demonstrate that miR-181a upregulation in the hippocampus promotes anxiety and ethanol-related behaviors, likely through SIRT-1 repression. This work highlights miR-181a as a key molecular mediator in the epigenetic regulation of mood disorders and ethanol consumption.

Depression and anxiety disorders are often exacerbated by social stress, necessitating the exploration of molecular mechanisms underlying stress resilience. Tissue plasminogen activator (tPA), a serine protease with pleiotropic effects in the brain, plays a critical role in modulating neuroplasticity and stress responses. This study investigates the behavioral and molecular effects of tPA gain-of-function in a social stress paradigm in male C57BL/6 mice using lentiviral vectors. Behaviorally, hippocampal tPA gain-of-function mitigated depression-like responses in the novelty-suppressed feeding, sucrose splash, tail suspension, and forced swim tests following exposure to chronic social stress. Additionally, in a two-bottle choice drinking paradigm, tPA overexpression reduced social stress-induced ethanol intake and preference, suggesting a role in dampening maladaptive coping behaviors. However, analysis of tastants’ intake and preference revealed no significant effects of tPA overexpression, indicating that it does not influence hedonic responses under stress conditions. Molecularly, tPA overexpression preserved hippocampal tPA mRNA expression and maintained levels of mature brain-derived neurotrophic factor in the hippocampus despite chronic stress exposure. These findings highlight the potential neuroprotective effects of tPA in maintaining hippocampal plasticity and mitigating stress-induced dysregulation of critical neurotrophic pathways. Collectively, this study underscores the potential of tPA as a therapeutic target for stress-induced mood and substance use disorders by modulating behavioral and neurobiological responses to chronic social stress.

Social isolation (SI) stress is increasingly recognized as a concern, associated with detrimental effects on mood and emotional well-being. Myelin Transcription Factor 1 (MyT1) is known for its pivotal role in nervous system development and mood regulation. This study delves into the potential of MyT1 to mitigate SI-induced behavioral abnormalities in mice. Utilizing a chronic SI model involving neonatal and post-weaning SI, male and female mice were subjected to lentiviral overexpression of MyT1 specifically in the medial prefrontal cortex (mPFC). A battery of behavioral assessments, including novelty-suppressed feeding, sucrose preference, sucrose splash, tape grooming, tail suspension, and forced swim tests, revealed notable antidepressant-like effects in both sexes upon MyT1 overexpression. Enhanced MyT1 expression corresponded with increased feeding initiation, sucrose preference, and self-grooming, alongside decreased immobility time. Importantly, the upregulation of MyT1 was accompanied by a significant reduction in cortical synaptotagmin 1 (Syt1) level. These findings underscore the involvement of MyT1 in mitigating SI-induced depression-like behavior. Moreover, the observed alterations in behavior are closely associated with changes in cortical Syt1 expression, suggesting its potential role as a target for unraveling the molecular mechanisms underlying mood disorders induced by SI. This study sheds light on the intricate interplay between MyT1 and cortical function in modulating responses to SI, paving the way for potential therapeutic interventions targeting these pathways.

Patients with stress-triggered major depression disorders (MDD) can often seek comfort or temporary relief through alcohol consumption, as they may turn to it as a means of self-medication or coping with overwhelming emotions. The use of alcohol as a coping mechanism for stressful events can escalate, fostering a cycle where the temporary relief it provides from depression can deepen into alcohol dependence, exacerbating both conditions. Although, the specific mechanisms involved in stress-triggered alcohol dependence and MDD comorbidities are not well understood, a large body of literature suggests that the serotonin transporter (SERT) plays a critical role in these abnormalities. To further investigate this hypothesis, we used a lentiviral-mediated knockdown approach to examine the role of hippocampal SERT knockdown in social defeat stress-elicited depression like behavior and ethanol-induced place preference (CPP). The results showed that social defeat stress-pro depressant effects were reversed following SERT knockdown demonstrated by increased sucrose preference, shorter latency to feed in the novelty suppressed feeding test, and decreased immobility time in the tail suspension and forced swim tests. Moreover, and most importantly, social stress-induced ethanol-CPP acquisition and reinstatement were significantly reduced following hippocampal SERT knockdown using short hairpin RNA shRNA-expressing lentiviral vectors. Finally, we confirmed that SERT hippocampal mRNA expression correlated with measures of depression- and ethanol-related behaviors by Pearson’s correlation analysis. Taken together, our data suggest that hippocampal serotoninergic system is involved in social stress-triggered mood disorders as well as in the acquisition and retrieval of ethanol contextual memory and that blockade of this transporter can decrease ethanol rewarding properties.

Emerging evidence suggests the serine protease, urokinase plasminogen activator (uPA), may play an important role in the modulation of mood and cognitive functions. Also, preliminary evidence indicates that uPA modulates BDNF activity that is known to be involved in the pathogenesis of mood disorders. However, the physiological functions of uPA in specific brain regions for mediating stress-related emotional behaviors remain to be elucidated. Therefore, the aim of this study was to assess the role of ectopic uPA expression on anxiety- and depression-like behaviors following social defeat stress in rats. For this purpose, we inspected the behavioral outcomes following bilateral stereotaxic delivery of uPA-overexpressing lentiviral vectors in the hippocampus using a series of behavioral tests. Results show that hippocampal uPA gain-of-function prevented stress-elicited anxiogenic-like effects, as determined in the marble burying, open field, and elevated plus maze tests, with no alterations in spontaneous locomotor activity. Also, ectopic uPA overexpression resulted in anti-depressant-like effects in the sucrose splash, tail suspension, and forced swim tests. Most importantly, uPA overexpression increased hippocampal BDNF levels, and a strong positive correlation was found using the Pearson test. Moreover, the same correlation analysis revealed a strong negative relationship between uPA mRNA and parameters of anxiety- and depression-like behaviors. Taken together, this work highlights the importance of considering uPA activation and provides new insights into the mechanisms involved in the pathophysiology of stress-elicited mood illnesses, which should help in the development of new approaches to tackle depression and anxiety disorders.

Epidemiological surveys have shown a strong relationship between maternal stress and offspring's mood disorders. Growing evidence suggested that environmental enrichment (EE) improves cognitive function in models of psychiatric and neurological disorders. However, the potential protective effects of gestational EE on social stress-elicited mood disorders in offspring have not been studied. Knowing that the undeveloped brain is more sensitive to gestational environmental stimuli, we hypothesized that initiating cognitive stimulation, during gestation, would protect against social stress-induced behavioral alterations in adulthood. Therefore, the present study aimed to investigate the effects of gestational EE on social stress-elicited anxiety- and ethanol-related behaviors in adult offspring. EE consisted of free access, of dams, to tubular devices of different shapes, colors, and sizes that were changed regularly. After birth and weaning, young adult offspring were exposed to 19 days of social stress and anxiety-like behavior was evaluated by elevated plus maze, open field, and marbles burying tests. The two-bottle choice (TBC) drinking paradigm was used to assess stress-induced ethanol intake. Results showed that gestational EE prevented social stress-elicited anxiogenic-like effects with no differences in spontaneous locomotor activity. Moreover, in the TBC paradigm, mice pre-exposed to EE consistently showed a significantly decreased consumption and preference for ethanol with no effects on tastants' intakes. Interestingly, gestational EE increased serum BDNF levels, which showed a correlation with measures of anxiety- and ethanolrelated behaviors. These findings indicate that some neurodevelopmental changes associated with prenatal EE may counteract adult social stress-induced behavioral alterations through a BDNF mechanism. Therefore, we propose that gestational EE has significant protective and beneficial effects on social stress-induced cognitive impairment. It can also alleviate anxiety-like behavior and subsequent excessive alcohol consumption.

The comorbidity of generalized anxiety disorders (GAD) with alcohol use disorders (AUD) is common and there is an association between the serotonin transporter (SERT) genetic variation and the comorbid conditions of GAD and AUD. However, few mechanistic studies have systematically explored the role of direct SERT manipulation in stress-elicited mood disorders. Therefore, the aim of this study was to determine whether reductions in SERT expression in the hippocampus were sufficient to ameliorate anxiety- and ethanol-related behaviors in socially defeated mice. Following stress exposure, and using stereotaxic surgery, SERT was knocked down using specific shRNA-expressing lentiviral vectors and anxiety-like behavior was evaluated by open-field, elevated plus maze, and marbles burying test. The two-bottle choice (TBC) drinking paradigm was used to assess stress-induced voluntary ethanol intake and preference. Results showed that hippocampal SERT loss-of-function prevented stress-elicited anxiogenic-like effects with no differences in spontaneous locomotor activity. Moreover, in the TBC paradigm, SERT shRNA-injected mice consistently showed a significantly decreased consumption and preference for ethanol when compared to Mock-injected controls. In contrast to ethanol, SERT shRNA-injected mice exhibited similar consumption and preference for saccharin and quinine. Interestingly, we confirmed that SERT hippocampal mRNA expression correlated with measures of anxiety- and ethanol-related behaviors by Pearson correlation analysis. Our findings show that social defeat recruits hippocampal serotoninergic system and that these neuroadaptations mediate the heightened anxiety-like behavior and voluntary alcohol intake observed following stress exposure, suggesting that this system represents a major brain stress element responsible for the negative reinforcement associated with the "dark side" of alcohol addiction.

Binge eating episodes are persistent and are essential features of numerous eating disorders (EDs). Susceptibility to EDs is largely presumed to be associated with early life stress. In fact, converging evidence from preclinical animal studies have implicated stress as a driver of binge eating. Still, literature examination indicates that vulnerability to EDs may depend on factors such as severity, time, and the type of stressor. Therefore, we aimed at exploring the link between chronic psychosocial stress and 'binge-like' sucrose intake in adolescent mice. To this aim, intruders' experimental mice were exposed to the chronic subordinate colony (CSC) housing, in the presence of a resident aggressive mouse for 2 weeks. At the end of the stress period, mice were tested for anxiety -like behavior then assessed for 'binge-like' intake of sucrose using a long-term drinking in the dark (DID) method that successfully replicates binge eating in humans. As expected, and compared to single housed colony controls (SHC), CSC exposure elicited an anxiogenic-like response in the open field (OF) and elevated-plus maze (EPM) tests and reduced weight gain. Most importantly, we report here for the first time, that mice exposed to chronic psychosocial stress displayed a 'binge-like' consumption of sucrose. However, neither quinine (bitter) nor saccharin (sweet) intakes were affected by CSC exposure. Finally, using Pearson's correlation, results showed a strong correlation between anxiety-like behavior parameters and sucrose intake. Overall these findings support the validity of our chronic psychosocial stress to model binge EDs and establish the long-term consequences of stress on 'binge-like' eating in male mice. These data suggest that chronic psychosocial stress is a risk factor for developing anxiety-associated EDs.

CD81, a member of the tetraspanin family, plays important roles in many physiological processes, such as cell motility, attachment, and entry. Yet, CD81 functions in the brain remain unclear. In this study, we investigated the effects of CD81 knockdown, using lentiviral vectors (LV), on anxiety- and ethanol-related behaviors. For this purpose, mice were stereotaxically injected with CD81 shRNA-expressing LV into the nucleus accumbens (Nacc) and were assessed for anxiety-like behavior using the elevated plus maze (EPM) and open field (OF) tests. Alcohol's sedative effects were studied using loss-of-righting-reflex (LORR) and voluntary ethanol intake was assessed using a two-bottle choice (TBC) procedure. Results showed that mice depleted of CD81 exhibited an anxiolytic-like response in the EPM and OF tests with no effect on locomotor activity. In addition, genetic reduction of CD81 in the Nacc increased mice' sensitivity to alcohol's sedative effects in the LORR test, although plasma alcohol concentrations were unaffected. Interestingly, CD81 loss-of-function-induced anxiolysis was accompanied by a significant decrease in ethanol, but not saccharin nor quinine, intake in the TBC procedure. Finally, and following CD81 mRNA quantification, Pearson's correlations showed a significant positive relationship between accumbal CD81 mRNA with anxiety and ethanol-related behaviors. Our data indicate that CD81 is implicated in the pathogenesis of anxiety and alcoholism. Indeed the targeted disruption of CD81, with the resultant decrease in CD81 mRNA in the Nacc, converted ethanol-"preferring" mice into ethanol "nonpreferring" mice. Collectively, these findings demonstrate that future CD81-targeted pharmacotherapies may be beneficial for the treatment of anxiety and alcoholism.

Rationale During the last few decades, alcohol use disorders (AUD) have reached an epidemic prevalence, yet social influences on alcoholism have not been fully addressed. Several factors can modulate alcohol intake. On one hand, stress can reinforce ethanol-induced behaviors and be an important component in AUD and alcoholism. On the other hand, environmental enrichment (EE) has a neuroprotective role and prevents the development of excessive ethanol intake in rodents. However, studies showing the role of EE in chronic psychosocial stress-impaired ethanol-conditioned rewards are nonexistent. Aim The purpose of the current study is to explore the potential protective role of EE on extinction and reinstatement of ethanol-conditioned place preference (EtOH-CPP) following chronic psychosocial stress. Methods In the first experiment and after the EtOH-CPP test, the mice were subjected to 15 days of chronic stress, then housed in a standard (SE) or enriched environment (EE) while EtOH-CPP extinction was achieved by repeated exposure to the CPP chambers without ethanol injection. In the second experiment and after the EtOH-CPP test, extinction was achieved as described above. Mice were then exposed to chronic stress for 2 weeks before being housed in a SE or EE. EtOH-CPP reinstatement was induced by a single exposure to the conditioning chambers. Results As expected, stress exposure increased anxiety-like behavior and reduced weight gain. More importantly, we found that EE significantly shortened chronic stress-delayed extinction and decreased the reinstatement of EtOH-CPP. Conclusion These results support the hypothesis that EE reduces the impact of alcohol-associated environmental stimuli, and hence it may be a general intervention for reducing cue-elicited craving and relapse in humans.

Recent studies have shown that Lethal-7 (let-7) microRNA (miRNA) is involved in a wide range of psychiatric disorders such as anxiety, depression, schizophrenia, and cocaine addiction. However, the exact role of let-7d miRNA in regulating ethanol intake and preference remains to be elucidated. The aim of the present study was to clarify the role of accumbal let-7d in controlling ethanol-related behaviors in adult rats. For this purpose, stereotaxic injections of let-7d-overexpressing lentiviral vectors (LV) were administered bilaterally into the nucleus accumbens (Nacc) of Wistar rats. The ethanol-related behaviors were investigated using the two-bottle choice (TBC) access paradigm, in which the rats had access to 2.5, 5, and 10% ethanol solutions, the grid hanging test (GHT) and ethanol-induced loss-of-righting-reflex (LORR) test. The results showed that intra-accumbally administered let-7d-overexpressing LV significantly decreased ethanol intake and preference without having significant effects on body weight, consumption or preference for tastants (saccharin and quinine) or ethanol metabolism. Furthermore, accumbal let-7d increased resistance to ethanol-induced sedation in the GHT and LORR test. Most importantly, the data showed that the dopamine D3 receptor (D3R) was a candidate target of let-7d In fact, and using real time PCR, let-7d was found to directly target D3R mRNA to decrease its expression. Further analyses proved that D3R expression was negatively correlated with the levels of let-7d and ethanol-related behaviors parameters. Taken together, the data indicating that let-7d impaired ethanol-related behaviors by targeting D3R will open up new exciting possibilities and might provide potential therapeutic evidence for alcoholism.

Alcoholism and alcohol use disorders are chronically relapsing conditions which is a major problem in treating alcohol addiction. In a previous study we showed that the dopamine transporter (DAT) is implicated in voluntary intake and preference. However, its role in modulating ethanol-associated contextual memory remains largely unknown. In this study we have investigated the role of DAT in ethanol-induced conditioned place preference (EtOH-CPP) acquisition and reinstatement in adult male C57BL/6 mice. For this purpose, we used both loss- and gain-of-function approaches to test the effects of central DAT manipulation on EtOH-CPP. We developed a lentiviral-mediated gene transfer approach to examine whether DAT knockdown (shDAT) or overexpression in the nucleus accumbens (Nacc) is enough to impair EtOH-CPP acquisition and reinstatement. In the first experiment, results showed that DAT knockdown blocked, whereas DAT overexpression, exacerbated the acquisition of EtOH-CPP. In the second experiment and after the EtOH-CPP expression, the mice were subjected to a 14-day extinction trials before drug-induced EtOH-CPP reinstatement was induced by a priming injection of 1 g/kg EtOH. Results indicated that reinstatement of EtOH-CPP was considerably decreased after accumbal shDAT injection. However, DAT overexpression significantly increased EtOH-CPP reinstatement. Finally, and following DAT mRNA quantification using RT-PCR, Pearson's correlation showed a strong positive relationship between accumbal DAT mRNA and EtOH-CPP acquisition and reinstatement. These results suggest that DAT expression in the Nacc is involved in the acquisition and retrieval of EtOH contextual memory and that blockade of this transporter can decrease the rewarding properties of EtOH.

RATIONALE:Dopamine levels are controlled in part by transport across the cell membrane by the dopamine transporter (DAT), and recent evidence showed that a polymorphism in the gene encoding DAT is associated with alcoholism. However, research in animal models using DAT knockout mice has yielded conflicting results.OBJECTIVES:The present study was planned to evaluate the effects of DAT knockdown in the nucleus accumbens (Nacc) on voluntary ethanol consumption and preference in male and female C57BL/6J mice.METHODS:For this purpose, animals were stereotaxically injected with DAT siRNA-expressing lentiviral vectors in the Nacc, and using a voluntary, continuous access two-bottle choice model of alcohol, we investigated the importance of accumbal DAT expression in voluntary alcohol intake and preference. We also investigated the effects of DAT knockdown on saccharin and quinine consumption and ethanol metabolism.RESULTS:We show that females consumed more alcohol than males. Interestingly, DAT knockdown in the Nacc significantly decreased alcohol intake and preference in both groups, but no significant sex by group interaction was observed. Also, DAT knockdown did not alter total fluid consumption, saccharin or quinine consumption, or blood ethanol concentrations. Using Pearson correlation, results indicated a strong positive relationship between DAT mRNA expression and ethanol consumption and preference.CONCLUSIONS:Taken together, these data provide further evidence that DAT plays an important role in controlling ethanol intake and that accumbal DAT contributes in the modulation of the reinforcing effects of ethanol. Overall, the results suggest that DAT inhibitors may be valuable in the pharmacotherapy of alcoholism.

Generalized anxiety and major depression disorders (MDD) are severe debilitating mood disorders whose etiology are not fully understood, but growing evidence indicates that microRNAs (miRNAs) might play a key role in their neuropathophysiological mechanisms. In the current study, we investigate the role of Lethal-7 (let-7d) miRNA, and its direct target dopamine D3 receptor (D3R) gain-of-function, in the hippocampus, in preclinical models of anxiety and depression in mice. For this purpose, we have constructed a lentiviral vector carrying let-7d miRNA and its anxiolytic effect was investigated by employing the open-field (OF) and the elevated plus maze (EPM) tests. The anti-depressant activity was evaluated using the tail suspension and the forced-swim tests (TST & FST). Our results show that let-7d overexpression significantly improved the measures of anxiety in the OF and EPM tests. In addition, let-7d increased the mobility time in the TST and FST. Interestingly, gene expression interaction analysis shows that the D3R mRNA negatively correlates with let-7d expression. In a different set of experiments, we used a tetracycline-inducible (tet-off) lentiviral vector to overexpress D3R to assess its gain-of-function in the hippocampus on anxiety- and depression-like behaviors. In line, we found that in the absence of doxycycline, D3R produced a significant anxiogenic and depressant-like response. Most importantly, these effects were abrogated when mice were fed doxycycline in drinking water. Our results provide the first evidence for an anxiolytic and anti-depressant-like action of let-7d through a potential D3R target-mediated mechanism which might open new avenues for anxiolytic and anti-depressant therapies.