Additive antinociceptive action of intrathecal anandamide reuptake inhibitor and morphine in the management of post-incisional pain in rats.

Spinal opioids have mixed efficacy and their adverse effects force treatment cessation of postoperative pain. Consequently, there is an ongoing search for new therapeutic strategies. Here, we evaluated the analgesic efficacy of intrathecal UCM707, an anandamide reuptake inhibitor, and morphine combination. Firstly, we assessed the effects of morphine (1, 5 and 10 µg), UCM707 (75 µg) and its combination in the hot plate. Then, morphine + UCM707 at sub-effective doses was evaluated in a rat post-incisional pain model. In addition, µ-, CB1r-, CB2r- and TRPV1-antagonists were pre-administered before the combination. Activation of µ-opioid and CB1r, and Cnr1, Cnr2, Oprm1 and TRPV1 expressions were evaluated in the lumbar sacra and periaqueductal grey by [35 S]-GTPγS binding autoradiography and qPCR studies. In the hot plate, morphine (1 µg) and UCM707 (75 µg) induced a more robust analgesic effect than each drug alone. Morphine plus UCM707 did not modify µ-opioid nor CB1 receptor function in the PAG or LS. Cnr1 and TRPV1 expression increased in the lumbar sacra (LS). Morphine plus UCM707 significantly reduced post-incisional pain at 1 and 4 days after surgery. Cnr1, Cnr2 and TRPV1 expressions increased in the LS. Blockade of µ-opioid receptor reduced combination effects on days 1 and 4. CB1r- and CB2r-antagonism reduced morphine + UCM707 effects on days 1 and 4, respectively. CB1r and TRPV1-antagonism improved their antinociceptive effects on day 4. These results revealed a synergistic/additive analgesic effect of UCM707 and morphine combination controlling postincisional pain. CB1r, CB2r and TRPV1 contribute differently as central sensitization occurs.

Cannabinoid Analgesia in Postoperative Pain Management: From Molecular Mechanisms to Clinical Reality.

Postoperative pain (POP) is a challenging clinical phenomenon that affects the majority of surgical patients and demands effective management to mitigate adverse outcomes such as persistent pain. The primary goal of POP management is to alleviate suffering and facilitate a seamless return to normal function for the patient. Despite compelling evidence of its drawbacks, opioid analgesia remains the basis of POP treatment. Novel therapeutic approaches rely on multimodal analgesia, integrating different pharmacological strategies to optimize efficacy while minimizing adverse effects. The recognition of the imperative role of the endocannabinoid system in pain regulation has prompted the investigation of cannabinoid compounds as a new therapeutic avenue. Cannabinoids may serve as adjuvants, enhancing the analgesic effects of other drugs and potentially replacing or at least reducing the dependence on other long-term analgesics in pain management. This narrative review succinctly summarizes pertinent information on the molecular mechanisms, clinical therapeutic benefits, and considerations associated with the plausible use of various cannabinoid compounds in treating POP. According to the available evidence, cannabinoid compounds modulate specific molecular mechanisms intimately involved in POP. However, only two of the eleven clinical trials that evaluated the efficacy of different cannabinoid interventions showed positive results.

Alterations of BDNF, mGluR5, Homer1a, p11 and excitatory/inhibitory balance in corticolimbic brain regions of suicide decedents.

BACKGROUND: Developing biological based approaches for preventing suicide has become a priority. In recent years, there has been a surge in studies investigating the role of the glutamatergic system in suicide, although it remains unclear. METHODS: We evaluated changes in the gene expression of the metabotropic glutamate receptor 5 (mGluR5) and its scaffolding proteins Homer1a and p11 in the dorsolateral prefrontal cortex (DLPFC), amygdala (AMY), and hippocampus (HIP) of 28 suicide decedents (S) (with no clinical psychiatric history or treatment with anxiolytics or antidepressants) and 26 controls (C) by real-time PCR (qPCR). Indeed, we measured BDNF gene expression and VGluT1 and VGAT immunoreactivities in the HIP by qPCR and immunohistochemistry, respectively. Cases and controls matched for age (C: 48.6 ± 11.6 years; S: 46.9 ± 14.5 years) and postmortem interval (PMI; C: 20.1 ± 13h; S: 16.9 ± 5h). RESULTS: In DLPFC, S had lower p11 gene expression levels, but no differences were found in mGluR5 or Homer1a. In the AMY and HIP, mGluR5 and Homer1a were increased, p11 and BDNF were reduced. In the HIP, there were less VGAT-ir and more VGluT1-ir. LIMITATIONS: Future studies are necessary to evaluate protein levels, and determine the cell types and potential compensatory mechanisms in a larger sample including S diagnosed with psychiatric disorders, females and different ethnicities. CONCLUSIONS: This study identified significant alterations in mGluR5, Homer1a, p11, BDNF and excitatory/inhibitory balance in corticolimbic brain areas of S. These results further characterize the biological basis of suicide, contributing to the identification of potential biomarkers for suicide prevention.

Biomarkers of the Endocannabinoid System in Substance Use Disorders.

Despite substance use disorders (SUD) being one of the leading causes of disability and mortality globally, available therapeutic approaches remain ineffective. The difficulty in accurately characterizing the neurobiological mechanisms involved with a purely qualitative diagnosis is an obstacle to improving the classification and treatment of SUD. In this regard, identifying central and peripheral biomarkers is essential to diagnosing the severity of drug dependence, monitoring therapeutic efficacy, predicting treatment response, and enhancing the development of safer and more effective pharmacological tools. In recent years, the crucial role that the endocannabinoid system (ECS) plays in regulating the reinforcing and motivational properties of drugs of abuse has been described. This has led to studies characterizing ECS alterations after exposure to various substances to identify biomarkers with potential diagnostic, prognostic, or therapeutic utility. This review aims to compile the primary evidence available from rodent and clinical studies on how the ECS components are modified in the context of different substance-related disorders, gathering data from genetic, molecular, functional, and neuroimaging experimental approaches. Finally, this report concludes that additional translational research is needed to further characterize the modifications of the ECS in the context of SUD, and their potential usefulness in the necessary search for biomarkers.

CB2 Receptor Involvement in the Treatment of Substance Use Disorders.

The pharmacological modulation of the cannabinoid receptor 2 (CB2r) has emerged as a promising potential therapeutic option in addiction. The purpose of this review was to determine the functional involvement of CB2r in the effects produced by drugs of abuse at the central nervous system (CNS) level by assessing evidence from preclinical and clinical studies. In rodents, several reports suggest the functional involvement of CB2r in the effects produced by drugs of abuse such as alcohol, cocaine, or nicotine. In addition, the discovery of CB2r in brain areas that are part of the reward system supports the relevance of CB2r in the field of addiction. Interestingly, animal studies support that the CB2r regulates anxiety and depression behavioral traits. Due to its frequent comorbidity with neuropsychiatric disorders, these pharmacological actions may be of great interest in managing SUD. Preliminary clinical trials are focused on exploring the therapeutic potential of modulating CB2r in treating addictive disorders. These promising results support the development of new pharmacological tools regulating the CB2r that may help to increase the therapeutic success in the management of SUD.

Cannabidiol: A Potential New Alternative for the Treatment of Anxiety, Depression, and Psychotic Disorders.

The potential therapeutic use of some Cannabis sativa plant compounds has been attracting great interest, especially for managing neuropsychiatric disorders due to the relative lack of efficacy of the current treatments. Numerous studies have been carried out using the main phytocannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD). CBD displays an interesting pharmacological profile without the potential for becoming a drug of abuse, unlike THC. In this review, we focused on the anxiolytic, antidepressant, and antipsychotic effects of CBD found in animal and human studies. In rodents, results suggest that the effects of CBD depend on the dose, the strain, the administration time course (acute vs. chronic), and the route of administration. In addition, certain key targets have been related with these CBD pharmacological actions, including cannabinoid receptors (CB1r and CB2r), 5-HT1A receptor and neurogenesis factors. Preliminary clinical trials also support the efficacy of CBD as an anxiolytic, antipsychotic, and antidepressant, and more importantly, a positive risk-benefit profile. These promising results support the development of large-scale studies to further evaluate CBD as a potential new drug for the treatment of these psychiatric disorders.

Association of cannabinoid receptor genes (CNR1 and CNR2) polymorphisms and panic disorder.

Background and objectives: Panic disorder (PD) is an anxiety disorder characterized by recurrent and unexpected panic attacks along with sudden onset of apprehension, fear or terror. The endocannabinoid system (ECS) has a role in stress recovery, regulating anxiety. The aim of this study was to analyze potential genetic alterations in key ECS targets in patients suffering from panic disorders.Design and methods: We analyzed single nucleotide polymorphisms (SNPs) of the cannabinoid receptors (CNR1; CNR2) and the endocannabinoid hydrolytic enzyme fatty acid amide hydrolase (FAAH) genes in 164 Spanish PD patients and 320 matched controls.Results: No significant differences were observed in the SNPs of the CNR2 and FAAH genes tested. However, when analyzing genotype-by-sex interaction at A592G (rs2501431) and C315T (rs2501432) in the CNR2 gene, the presence of the G-allele in males was associated with a protective haplotype. Genotyping analysis revealed that variants in CNR1 confer vulnerability to PD, with a significantly increased risk associated with the G-allele (rs12720071) and C-allele (rs806368). This finding was consistent when analyzing genotype-by-sex interaction, where females presented a greater PD risk.Conclusions: Polymorphisms at the CNR1 gene may be a risk factor for PD contributing to sex-specific dysfunction in females.

Gender differences in the effects of cannabidiol on ethanol binge drinking in mice.

The purpose of this study was to explore the effects of cannabidiol (CBD) on binge drinking and evaluate potential gender-related differences. To this aim, male and female C57BL/6J mice (n = 60 per sex) were exposed to the drinking in the dark (DID) model for 4 weeks (DID-1 to DID-4). Dose-response effects of CBD on the ethanol intake were tested by acute (day-4 of DID-3) or repeated administration (day-1 to 4 of DID-4) (experiment 1: CBD 15, 30, and 60 mg/kg, i.p.; experiment 2: CBD 90 mg/kg, i.p.). Finally, we analyzed the relative gene expression of tyrosine hydroxylase (TH) and µ-opioid receptor (OPRM1) and cannabinoid CB1 receptor (CB1 r) in the ventral tegmental area (VTA) and in the nucleus accumbens (NAc), respectively, by real-time quantitative PCR. Females exhibited higher ethanol intake during each DID session. Interestingly, females also showed higher expression of TH and OPRM1, without any difference in CB1 r. Only the acute administration of CBD at the highest dose (90 mg/kg) reduced significantly ethanol consumption in both sexes. Chronic CBD administration (30, 60 and 90 mg/kg) reduced ethanol intake in males, whereas in females a significant reduction was only achieved with the highest dose (90 mg/kg). Repeated administration with CBD (60 mg/kg) significantly reduced TH and OPRM1 in males. In addition, CBD (30 and 60 mg/kg) significantly reduced CB1 r in males. No effect was observed in females. Taken together, these findings suggest that CBD may be of interest for treating binge-drinking patterns and that gender-related difference may affect the treatment outcome.

Cannabidiol does not display drug abuse potential in mice behavior.

Recent evidence suggests that cannabidiol (CBD) may be useful for the treatment of different neuropsychiatric disorders. However, some controversy regarding its profile as a drug of abuse hampers the further development of basic and clinical studies. In this study, the behavioral profile of CBD as a potential drug of abuse was evaluated in C57BL/6J mice. Reinforcing properties of CBD (15, 30, and 60 mg/kg; i.p.) were assessed using the conditioned place preference (CPP) paradigm. Spontaneous withdrawal symptoms and motor activity in the open field were examined 12 h after the last CBD administration (30 mg/kg/12 h, i.p., 6 days). CBD plasma concentrations were measured at 2, 4, 8, 12, and 24 h after the administration of CBD (30 mg/kg, i.p.). Furthermore, an oral CBD self-administration paradigm (50 mg/kg; CBD water-soluble 1.2 mg/mL) was performed to evaluate whether this drug produced any effects on motivation compared with a non-reinforcing substance (water). We found that CBD failed to induce CPP, withdrawal symptoms, or altered motor behavior 12 h after its administration. At that time, only traces of CBD were detected, ensuring that the lack of alterations in somatic signs and locomotor activity was not due to residual drug in plasma. Interestingly, mice displayed similar motivation and consumption of CBD and water. Taken together, these results show that CBD lacks activity as a drug of abuse and should stimulate the development of the basic and clinical studies needed to elucidate its potential therapeutic use for the treatment of neuropsychiatric and drug use disorders.

Cannabidiol regulates the expression of hypothalamus-pituitary-adrenal axis-related genes in response to acute restraint stress.

BACKGROUND: Research interest has grown around the potential therapeutic use of cannabidiol in mood-related disorders, due to its anxiolytic and antidepressant-like effects. These have been partially attributed to its action as an allosteric modulator of 5-HTR1A. However, the exact mechanism supporting cannabidiol properties remains unclear. AIMS: To assess the effects of cannabidiol on different targets of the hypothalamus-pituitary-adrenal axis under baseline and stress conditions. METHODS: We administered cannabidiol (5 mg/kg, 15 mg/kg or 30 mg/kg, intraperitoneally) or vehicle to male C57BL/6J mice 90 min before single restraint stress exposure (20 min). Using real-time polymerase chain reaction analysis, we measured alterations in the relative gene expression of corticotropin-releasing factor in the paraventricular nucleus, pro-opiomelanocortin in the arcuate nucleus of the hypothalamus, glucocorticoid receptor in the hippocampus, and serotonin 5-HTR1A receptor in the hippocampus and amygdala. RESULTS: Under baseline conditions, cannabidiol did not modify any element of the hypothalamus-pituitary-adrenal axis. In contrast, all doses induced alterations in 5-HTR1A in the amygdala and hippocampus. Interestingly, cannabidiol at low (5 mg/kg) and intermediate doses (15 mg/kg) successfully blocked the effects induced by acute stress on corticotropin-releasing factor, pro-opiomelanocortin and glucocorticoid receptor gene expression. Also, restraint stress induced the opposite effects in 5-HTR1A gene expression in the hippocampus and amygdala, an effect not seen in mice treated with cannabidiol at low doses. CONCLUSIONS: Taken together, these data suggest the ability of cannabidiol to regulate acute stress hypothalamus-pituitary-adrenal axis activation might be explained, at least in part, by its action on 5-HTR1A receptors.

Deletion of Dlk1 increases the vulnerability to developing anxiety-like behaviors and ethanol consumption in mice.

Anxiety and alcohol use disorders (AUD) often present together, constituting a significant public health problem worldwide. In this study, we investigated the role of DLK1, a ligand of the Delta/NOTCH epidermal growth factor (EGF)-like protein family, reported to play a role in DA neurons differentiation in the striatum, as a neurobiological factor involved in the mechanisms regulating this psychiatric comorbidity. We exposed Dlk1 knockout mice (Dlk1-/- mice) to the open-field (OF), the light-dark box (LBD) and the elevated plus maze (EPM) tests, evaluating motivation to drink and ethanol consumption using the oral ethanol self-administration (OEA) paradigm. Quantitative real time polymerase chain reaction (qPCR) studies were carried out to evaluate alterations in targets closely related to DA neurotransmission in the reward system, tyrosine hydroxylase (Th) in the ventral tegmental area (VTA), and µ-opioid receptor (Oprm1) in the nucleus accumbens (NAc). No differences were observed in the total or peripheral distances travelled by Dlk1-/- compared to wild-type (WT) mice in OF. However, central distance travelled significantly decreased in Dlk1-/- mice. Deletion of Dlk1 increased anxiety-like behaviors in the LDB and EPM, and, Dlk1-/- mice also presented higher ethanol intake and motivation to drink (number of effective responses) in the OEA. In addition, Th and Oprm1 gene expression was reduced in the VTA and NAc of Dlk1-/- mice. We conclude that deletion of Dlk1 increases anxiety-related behaviors and vulnerability to ethanol consumption and modifies the gene expression of key targets closely related with DA neurotransmission involved in the reinforcing actions of ethanol.

Role of the endocannabinoid system in drug addiction.

Drug addiction is a chronic relapsing disorder that produces a dramaticglobal health burden worldwide. Not effective treatment of drug addiction is currently available probably due to the difficulties to find an appropriate target to manage this complex disease raising the needs for further identification of novel therapeutic approaches. The endocannabinoid system has been found to play a crucial role in the neurobiological substrate underlying drug addiction. Endocannabinoids and cannabinoid receptors are widely expressed in the main areas of the mesocorticolimbic system that participate in the initiation and maintenance of drug consumption and in the development of compulsion and loss of behavioral control occurring during drug addiction. The identification of the important role played by CB1 cannabinoid receptors in drug addiction encouraged the possible used of an early commercialized CB1 receptor antagonist for treating drug addiction. However, the incidence of serious psychiatric adverse events leaded to the sudden withdrawal from the market of this CB1 antagonist and all the research programs developed by pharmaceutical companies to obtain new CB1 antagonists were stopped. Currently, new research strategies are under development to target the endocannabinoid system for drug addiction avoiding these side effects, which include allosteric negative modulators of CB1 receptors and compounds targeting CB2 receptors. Recent studies showing the potential role of CB2 receptors in the addictive properties of different drugs of abuse have open a promising research opportunity to develop novel possible therapeutic approaches.

Effects of cannabidiol plus naltrexone on motivation and ethanol consumption.

BACKGROUND AND PURPOSE: The aim of this study was to explore if the administration of naltrexone together with cannabidiol (CBD) may improve the efficacy in reducing alcohol consumption and motivation rather than any of the drugs given separately. EXPERIMENTAL APPROACH: The effects of low doses of naltrexone (0.7 mg·kg-1 , p.o.) and/or CBD (20 mg·kg-1 ·day-1 , s.c.) on ethanol consumption and motivation to drink were evaluated in the oral-ethanol self-administration paradigm in C57BL/6 mice. Gene expression analyses of the opioid µ receptor (Oprm1) in the nucleus accumbens (NAc), tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) and the 5-HT1A receptor in the dorsal raphe nucleus (DR) were carried out by real-time PCR. The role of 5-HT1A receptors in the ethanol reduction induced by the administration of CBD + naltrexone was analysed by using the 5-HT1A receptor antagonist WAY100635 (0.3 mg·kg-1 , i.p.). KEY RESULTS: The administration of CBD + naltrexone significantly reduced motivation and ethanol intake in the oral self-administration procedure in a greater proportion than the drugs given alone. Only the combination of both drugs significantly reduced Oprm1, TH and 5-HT1A gene expressions in the NAc, VTA and DR respectively. Interestingly, the administration of WAY100635 significantly blocked the actions of CBD + naltrexone but had no effects by itself. CONCLUSION AND IMPLICATIONS: The combination of low doses of CBD plus naltrexone were more effective than either CBD or naltrexone alone at reducing ethanol consumption and the motivation to drink. These effects appear to be mediated, at least in part, by 5-HT1A receptors.

Alterations in Gene and Protein Expression of Cannabinoid CB2 and GPR55 Receptors in the Dorsolateral Prefrontal Cortex of Suicide Victims.

Recent studies point to the cannabinoid CB2 receptors (CB2r) and the non-cannabinoid receptor GPR55 as potential key targets involved in the response to stress, anxiety, and depression. Considering the close relationship between neuropsychiatric disorders and suicide, the purpose of this study was to evaluate the potential alterations of CB2r and GPR55 in suicide victims. We analyzed gene and protein expression of both receptors by real-time PCR and western blot, respectively, in the dorsolateral prefrontal cortex (DLPFC) of 18 suicide victims with no clinical psychiatric history or treatment with anxiolytics or antidepressants, and 15 corresponding controls. We used in situ proximity ligation assay to evaluate whether the receptors formed heteromeric complexes and to determine the expression level of these heteromers, also assessing the co-expression of heteromers in neurons, astroglia, or microglia cells. CB2r and GPR55 gene expressions were significantly lower (by 33 and 41%, respectively) in the DLPFC of suicide cases. CB2r protein expression was higher, as were CB2-GPR55 heteroreceptor complexes. The results also revealed the presence of CB2-GPR55 receptor heteromers in both neurons and astrocytes, whereas microglial cells showed no expression. We did not observe any significant alterations of GPR55 protein expression. Additional studies will be necessary to evaluate if these alterations are reproducible in suicide victims diagnosed with different psychiatric disorders. Taken together, the results suggest that CB2r and GPR55 may play a relevant role in the neurobiology of suicide.

Cannabidiol reduces ethanol consumption, motivation and relapse in mice.

This study evaluated the effects of cannabidiol (CBD) on ethanol reinforcement, motivation and relapse in C57BL/6 J mice. The effects of CBD (60 mg/kg, i.p.) on blood ethanol concentration, hypothermia and handling-induced convulsions associated to acute ethanol administration were evaluated. The two-bottle choice paradigm was performed to assess the effects of CBD (30, 60 and 120 mg/kg/day, i.p.) on ethanol intake and preference. In addition, an oral ethanol self-administration experiment was carried out to evaluate the effects of CBD [a single s.c. administration of a microparticle formulation providing CBD continuous controlled release (30 mg/kg/day)] on the reinforcement and motivation for ethanol. The effects of CBD (60 and 120 mg/kg/day, i.p.) on ethanol-induced relapse were also evaluated. Gene expression analyses of tyrosine hydroxylase in ventral tegmental area and µ-opioid (Oprm1), cannabinoid (CB1 r and CB2 r) and GPR55 receptors in nucleus accumbens (NAcc) were carried out by real-time polymerase chain reaction. Cannabidiol reduced the ethanol-induced hypothermia and handling-induced convulsion but failed to modify blood ethanol concentration. CBD reduced ethanol consumption and preference in the two-bottle choice, significantly decreased ethanol intake and the number of effective responses in the oral ethanol self-administration, and reduced ethanol-induced relapse. Furthermore, the administration of CBD significantly reduced relative gene expression of tyrosine hydroxylase in the ventral tegmental area, Oprm1, CB1 r and GPR55 in the NAcc and significantly increased CB2 r in the NAcc. Taken together, these results reveal that the administration of CBD reduced the reinforcing properties, motivation and relapse for ethanol. These findings strongly suggest that CBD may result useful for the treatment of alcohol use disorders.

Deletion of Dlk2 increases the vulnerability to anxiety-like behaviors and impairs the anxiolytic action of alprazolam.

The purpose of this study was to evaluate the role of the non-canonical DLK2 NOTCH ligand in the regulation of emotional behavior. To this aim, anxiety and depressive-like behaviors were examined in Dlk2 knock-out (Dlk2-/-) and its corresponding wild-type (WT) mice. Furthermore, relative gene expression analyses of corticotropin releasing hormone (Crh) in the paraventricular nucleus (PVN), glucocorticoid receptor (NR3C1) and FK506 binding protein 5 (FKBP5) in the hippocampus (HIPP), and the transcription factors Hes1, Hes5 and Hey1 in the PVN, HIPP and amygdala (AMY) were carried out in Dlk2-/- and WT mice under basal conditions and after exposure to restraint stress. The anxiolytic action of alprazolam and the relative gene expression levels of the GABA-A alpha 2 and gamma 2 receptor subunits (Gabra2 and Gabrg2) were also evaluated in the HIPP and AMY of WT and Dlk2-/- mice. The results reveal that deletion of Dlk2 increased anxiety and depressive-like behaviors and altered the vulnerability to restraint stress on Crh gene expression in the PVN, Nr3c1 and Fkbp5 gene expression in the HIPP, and Hes1, Hes5 and Hey1 gene expression in the PVN, HIPP and AMY. Interestingly, the administration of alprazolam failed to produce an anxiolytic action in Dlk2-/- mice. Indeed, Gabra2 and Gabrg2 gene expression levels were significantly affected under basal conditions and after stress exposure in Dlk2-/- mice compared with WT mice. In conclusion, the results suggest that DLK2 plays an important role in the regulation of emotional behaviors and relevant targets of the stress axis, NOTCH pathway and GABAergic neurotransmission. In addition, the deletion of Dlk2 blocked the anxiolytic response to alprazolam. Future studies are needed to determine the relevance of DLK2 as a potential therapeutic target for the treatment of neuropsychiatric disorders with anxiety or depressive-like behaviors.

Increased vulnerability to ethanol consumption in adolescent maternal separated mice.

The purpose of this study was to evaluate the effects of early life stress on the vulnerability to ethanol consumption in adolescence. To this aim, mice were separated from their mothers for 12 hours/day on postnatal days 8 and 12. Emotional behavior (light-dark box, elevated plus maze and tail suspension tests) and pre-attentional deficit (pre-pulse inhibition) were evaluated in adolescent maternal separated (MS) mice. Alterations of the corticotropin-releasing factor (CRF), glucocorticoid receptor (NR3C1), tyrosine hydroxylase (TH), mu-opioid receptor (MOr), brain-derived neurotrophic factor (BDNF), neuronal nuclei (NeuN), microtubule-associated protein 2 (MAP2) and neurofilament heavy (NF200)-immunoreactive fibers were studied in the paraventricular nucleus of the hypothalamus (PVN), ventral tegmental area (VTA), nucleus accumbens (NAc) or hippocampus (HIP). The effects of maternal separation (alone or in combination with additional stressful stimuli) on ethanol consumption during adolescence were evaluated using the oral ethanol self-administration paradigm. MS mice presented mood-related alterations and pre-attentional deficit. Increased CRF, MOr and TH, and reduced BDNF, NR3C1, NeuN, MAP2 and NF200-immunoreactive fibers were observed in the PVN, NAc and HIP of adolescent MS mice. In the oral ethanol self-administration test, adolescent MS mice presented higher ethanol consumption and motivation. Exposure to additional new stressful stimuli during adolescence significantly increased the vulnerability to ethanol consumption induced by maternal separation. These results clearly demonstrated that exposure to early life stress increased the vulnerability to ethanol consumption, potentiated the effects of stressful stimuli exposure during adolescence on ethanol consumption and modified the expression of key targets involved in the response to stress, ethanol reinforcing properties and cognitive processes.