N-Acetylcysteine normalizes brain oxidative stress and neuroinflammation observed after protracted ethanol abstinence: a preclinical study in long-term ethanol-experienced male rats.

RATIONALE: Using a preclinical model based on the Alcohol Deprivation Effect (ADE), we have reported that N-Acetylcysteine (NAC) can prevent the relapse-like drinking behaviour in long-term ethanol-experienced male rats. OBJECTIVES: To investigate if chronic ethanol intake and protracted abstinence affect several glutamate transporters and whether NAC, administered during the withdrawal period, could restore the ethanol-induced brain potential dysfunctions. Furthermore, the antioxidant and anti-inflammatory effects of NAC during abstinence in rats under the ADE paradigm were also explored. METHODS: The expression of GLT1, GLAST and xCT in nucleus accumbens (Nacc) and dorsal striatum (DS) of male Wistar was analysed after water and chronic ethanol intake. We used the model based on the ADE within another cohort of male Wistar rats. During the fourth abstinence period, rats were treated for 9 days with vehicle or NAC (60, 100 mg/kg; s.c.). The effects of NAC treatment on (i) glutamate transporters expression in the Nacc and DS, (ii) the oxidative status in the hippocampus (Hip) and amygdala (AMG) and (iii) some neuroinflammatory markers in prefrontal cortex (PFC) were tested. RESULTS: NAC chronic administration during protracted abstinence restored oxidative stress markers (GSSG and GGSH/GSH) in the Hip. Furthermore, NAC was able to normalize some neuroinflammation markers in PFC without normalizing the observed downregulation of GLT1 and GLAST in Nacc. CONCLUSIONS: NAC restores brain oxidative stress and neuroinflammation that we previously observed after protracted ethanol abstinence in long-term ethanol-experienced male rats. This NAC effect could be a plausible mechanism for its anti-relapse effect. Also, brain oxidative stress and neuroinflammation could represent and identify plausible targets for searching new anti-relapse pharmacotherapies.

Crosstalk between Mu-Opioid receptors and neuroinflammation: Consequences for drug addiction and pain.

Mu-Opioid Receptors (MORs) are well-known for participating in analgesia, sedation, drug addiction, and other physiological functions. Although MORs have been related to neuroinflammation their biological mechanism remains unclear. It is suggested that MORs work alongside Toll-Like Receptors to enhance the release of pro-inflammatory mediators and cytokines during pathological conditions. Some cytokines, including TNF-α, IL-1ß and IL-6, have been postulated to regulate MORs levels by both avoiding MOR recycling and enhancing its production. In addition, Neurokinin-1 Receptor, also affected during neuroinflammation, could be regulating MOR trafficking. Therefore, inflammation in the central nervous system seems to be associated with altered/increased MORs expression, which might regulate harmful processes, such as drug addiction and pain. Here, we provide a critical evaluation on MORs' role during neuroinflammation and its implication for these conditions. Understanding MORs' functioning, their regulation and implications on drug addiction and pain may help elucidate their potential therapeutic use against these pathological conditions and associated disorders.

Neuroimmune and Mu-Opioid Receptor Alterations in the Mesocorticolimbic System in a Sex-Dependent Inflammatory Pain-Induced Alcohol Relapse-Like Rat Model.

Evidence concerning the role of alcohol-induced neuroinflammation in alcohol intake and relapse has increased in the last few years. It is also proven that mu-opioid receptors (MORs) mediate the reinforcing properties of alcohol and, interestingly, previous research suggests that neuroinflammation and MORs could be related. Our objective is to study neuroinflammatory states and microglial activation, together with adaptations on MOR expression in the mesocorticolimbic system (MCLS) during the abstinence and relapse phases. To do so, we have used a sex-dependent rat model of complete Freund's adjuvant (CFA)-induced alcohol deprivation effect (ADE). Firstly, our results confirm that only CFA-treated female rats, the only experimental group that showed relapse-like behavior, exhibited specific alterations in the expression of phosphorylated NFκB, iNOS, and COX2 in the PFC and VTA. More interestingly, the analysis of the IBA1 expression revealed a decrease of the microglial activation in PFC during abstinence and an increase of its expression in the relapse phase, together with an augmentation of this activation in the NAc in both phases that only occur in female CFA-treated rats. Additionally, the expression of IL1ß also evidenced these dynamic changes through these two phases following similar expression patterns in both areas. Furthermore, the expression of the cytokine IL10 showed a different profile than that of IL1ß, indicating anti-inflammatory processes occurring only during abstinence in the PFC of CFA-female rats but neither during the reintroduction phase in PFC nor in the NAc. These data indicate a downregulation of microglial activation and pro-inflammatory processes during abstinence in the PFC, whereas an upregulation can be observed in the NAc during abstinence that is maintained during the reintroduction phase only in CFA-female rats. Secondly, our data reveal a correlation between the alterations observed in IL1ß, IBA1 levels, and MOR levels in the PFC and NAc of CFA-treated female rats. Although premature, our data suggest that neuroinflammatory processes, together with neural adaptations involving MOR, might play an important role in alcohol relapse in female rats, so further investigations are warranted.

The Effects of N-Acetylcysteine on the Rat Mesocorticolimbic Pathway: Role of mGluR5 Receptors and Interaction with Ethanol.

N-acetylcysteine (NAC) is a prodrug that is marketed as a mucolytic agent and used for the treatment of acetaminophen overdose. Over the last few decades, evidence has been gathered that suggests the potential use of NAC as a new pharmacotherapy for alcohol use disorder (AUD), although its mechanism of action is already being debated. In this paper, we set out to assess both the potential involvement of the glutamate metabotropic receptors (mGluR) in the possible dual effect of NAC administered at two different doses and NAC's effect on ethanol-induced activation. To this aim, 30 or 120 mg/kg of NAC was intraperitoneally administered to rats with the presence or absence of the negative allosteric modulator of mGluR5 (MTEP 0.1 mg/kg). Thereafter, the cFOS IR-cell expression was analyzed. Secondly, we explored the effect of 120 mg/kg of NAC on the neurochemical and behavioral activation induced by intra-VTA ethanol administration (150 nmol). Our results showed that the high NAC dose stimulated cFOS expression in the NAcc, and that this effect was suppressed in the presence of MTEP, thus suggesting the implication of mGluR5. Additionally, high doses could attenuate the ethanol-induced increase in cFOS-expression in the NAcc, probably due to a phenomenon based on the long-term depression of the MSNs. Additional experiments are required to corroborate our hypothesis.

Efficacy of N-acetylcysteine in the prevention of alcohol relapse-like drinking: Study in long-term ethanol-experienced male rats.

Alcohol use disorders are chronic and highly relapsing disorders, thus alcoholic patients have a high rate of recidivism for drug use even after long periods of abstinence. The literature points to the potential usefulness of N-acetylcysteine (NAC) in the management of several substance use disorders probably due to its capacity to restore brain homeostasis of the glutamate system disrupted in addiction. However, there is little evidence in the case of alcohol. The aim of this study was to explore the potential anti-relapse efficacy of NAC using the alcohol deprivation effect (ADE) model in long-term experienced rats. Two experiments were performed in male Wistar rats to: (a) test the efficacy of NAC to prevent relapse and (b) discriminate the best administration schedule (intermittent vs. continuous) for NAC. In the first experiment, animals were implanted with mini-osmotic pumps delivering 0 or 1 mg/hr NAC during 14 days. In a second experiment, rats received 0, 60, or 100 mg/kg once daily by subcutaneous injection. The efficacy to prevent ADE was evaluated in both experiments. NAC subcutaneously administered, either by continuous infusion or by intermittent injections regimen, is able to block the ADE. The best results were obtained after using 60 mg/kg NAC dose. Our findings support the hypothesis that NAC may represent a valuable therapy in the management of alcohol relapse.

Impaired alcohol-induced dopamine release in the nucleus accumbens in an inflammatory pain model: behavioral implications in male rats.

ABSTRACT: Recent studies have drawn the attention to the link between alcohol use disorder and the presence of pain. Indeed, the correct management of pain in patients with a previous history of alcohol use disorder has been reported to decrease the risk of relapse in alcohol drinking, suggesting that in this prone population, pain may increase the vulnerability to relapse. Previous data in male rats revealed that inflammatory pain desensitizes mu-opioid receptors in the ventral tegmental area and increases intake of high doses of heroin. Owing to the relevant role of mu-opioid receptors in alcohol effects, we hypothesize that pain may also alter alcohol reinforcing properties and therefore affect alcohol relapse in male rats. Our microdialysis studies show that the presence of inflammatory pain blunted the increase of extracellular dopamine levels in the nucleus accumbens induced by 1.5 g/kg of ethanol (s.c.). Moreover, we also revealed that the administration of 52 nmol of ethanol into the ventral tegmental area failed to induce place preference only in inflammatory pain-suffering animals, and a higher dose (70 nmol) was necessary to reverse this effect. Finally, we evaluated the effect of inflammatory pain on the alcohol deprivation effect in long-term ethanol-experienced male rats. After 4 cycles of free ethanol intake and abstinence periods, inflammatory pain induced alcohol deprivation effect without affecting its magnitude. These intriguing data reveal the impact of pain on neurochemical and behavioral effects after alcohol administration but also underscore the necessity of finding an appropriate paradigm to determine the long-term behavioral consequences.

Dose-dependent induction of CPP or CPA by intra-pVTA ethanol: Role of mu opioid receptors and effects on NMDA receptors.

The neurobiological mechanisms underlying alcohol motivational properties are still not fully understood, however, the mu-opioid receptors (MORs) have been evidenced as central elements in the manifestation of the alcohol reinforcing properties. Drug-associated environmental stimuli can trigger alcohol relapse and promote alcohol consumption whereby N-methyl-d-aspartate (NMDA) receptors play a pivotal role. Here we sought to demonstrate, for the first time, that ethanol induces conditioned place preference or aversion (CPP or CPA) when administered locally into the ventral tegmental area (VTA) and the associated role of MORs. We further analyzed the changes in the expression and mRNA levels of GluN1 and GluN2A subunits in designated brain areas. The expression of CPP or CPA was characterized following intra-VTA ethanol administration and we showed that either reinforcing (CPP) or aversive (CPA) properties are dependent on the dose administered (ranging here from 35 to 300 nmol). Furthermore, the critical contribution of local MORs in the acquisition of CPP was revealed by a selective antagonist, namely ß-Funaltrexamine. Finally, modifications of the expression of NMDA receptor subunits in the Nucleus Accumbens (NAc) and Hippocampus after ethanol-induced CPP were analyzed at the proteomic and transcriptomic levels by western blot and In Situ Hybridation RNAscope techniques, respectively. Results showed that the mRNA levels of GluN2A but not GluN1 in NAc are higher after ethanol CPP. These novel results pave the way for further characterisation of the mechanisms by which ethanol motivational properties are associated with learned environmental cues.

Activation of MORs in the VTA induces changes on cFos expression in different projecting regions: Effect of inflammatory pain.

Chronic pain is a worldwide major health problem and many pain-suffering patients are under opioid based therapy. Epidemiological data show that pain intensity correlates with the risk of misuse of prescription opioids, and other drugs of abuse including alcohol. This increased vulnerability to suffer Substance Use Disorders could be, in part, caused by functional changes that occur over the mesocorticolimbic system, a brain pathway involved in reward processing and addiction. Previous data in rats revealed that inflammatory pain desensitizes mu opioid receptors (MORs) in the ventral tegmental area (VTA). As a consequence, pain alters dopamine release in the nucleus accumbens (NAc) derived from MOR activation in the VTA and also increases intake of high doses of heroine. Given that the VTA neurons target different brain regions, in the present study we first analyzed changes induced by inflammatory pain in the MOR dependent activation pattern of the main VTA projecting areas. To do that, we administered two doses (7 or 14 ng) of DAMGO (MORs agonist) or artificial cerebrospinal fluid (aCSF) focally into the VTA of rats and measured the activation in projection areas by cFos immunohistochemistry. Our results show that focal injections of DAMGO in the VTA increases cFos expression in the majority of its projecting areas, namely NAc, basolateral amygdala (BLA), cingulate cortex (ACC) and bed nucleus of the stria terminalis (BNST), as compared to aCSF. Second, we analyzed whether inflammatory pain would affect to cFos expression using a group of rats injected with CFA in the hind paw. In this case, we found that cFos expression was not significantly different between DAMGO and aCSF administered rats in BLA, ACC and BNST. Our results confirm that inflammatory pain induces desensitization of VTA MORs in a region dependent manner which can be very relevant for addictive behaviours.

Mystic Acetaldehyde: The Never-Ending Story on Alcoholism.

After decades of uncertainties and drawbacks, the study on the role and significance of acetaldehyde in the effects of ethanol seemed to have found its main paths. Accordingly, the effects of acetaldehyde, after its systemic or central administration and as obtained following ethanol metabolism, looked as they were extensively characterized. However, almost 5 years after this research appeared at its highest momentum, the investigations on this topic have been revitalized on at least three main directions: (1) the role and the behavioral significance of acetaldehyde in different phases of ethanol self-administration and in voluntary ethanol consumption; (2) the distinction, in the central effects of ethanol, between those arising from its non-metabolized fraction and those attributable to ethanol-derived acetaldehyde; and (3) the role of the acetaldehyde-dopamine condensation product, salsolinol. The present review article aims at presenting and discussing prospectively the most recent data accumulated following these three research pathways on this never-ending story in order to offer the most up-to-date synoptic critical view on such still unresolved and exciting topic.

Pre-Clinical Studies with D-Penicillamine as a Novel Pharmacological Strategy to Treat Alcoholism: Updated Evidences.

Ethanol, as other drugs of abuse, is able to activate the ventral tegmental area dopamine (VTA-DA) neurons leading to positively motivational alcohol-seeking behavior and use, and, ultimately to ethanol addiction. In the last decades, the involvement of brain-derived acetaldehyde (ACD) in the ethanol actions in the mesolimbic pathway has been widely demonstrated. Consistent published results have provided a mechanistic support to the use of ACD inactivating agents to block the motivational and reinforcing properties of ethanol. Hence, in the last years, several pre-clinical studies have been performed in order to analyze the effects of the sequestering ACD agents in the prevention of ethanol relapse-like drinking behavior as well as in chronic alcohol consumption. In this sense, one of the most explored interventions has been the administration of D-Penicillamine (DP). These pre-clinical studies, that we critically summarize in this article, are considered a critical step for the potential development of a novel pharmacotherapeutic strategy for alcohol addiction treatment that could improve the outcomes of current ones. Thus, on one hand, several experimental findings provide the rationale for using DP as a novel therapeutic intervention alone and/or in combination to prevent relapse into alcohol seeking and consumption. On the other hand, its effectiveness in reducing voluntary ethanol consumption in long-term experienced animals still remains unclear. Finally, this drug offers the additional advantage that has already been approved for use in humans, hence it could be easily implemented as a new therapeutic intervention for relapse prevention in alcoholism.

Glutamate and Opioid Antagonists Modulate Dopamine Levels Evoked by Innately Attractive Male Chemosignals in the Nucleus Accumbens of Female Rats.

Sexual chemosignals detected by vomeronasal and olfactory systems mediate intersexual attraction in rodents, and act as a natural reinforcer to them. The mesolimbic pathway processes natural rewards, and the nucleus accumbens receives olfactory information via glutamatergic projections from the amygdala. Thus, the aim of this study was to investigate the involvement of the mesolimbic pathway in the attraction toward sexual chemosignals. Our data show that female rats with no previous experience with males or their chemosignals display an innate preference for male-soiled bedding. Focal administration of the opioid antagonist ß-funaltrexamine into the posterior ventral tegmental area does not affect preference for male chemosignals. Nevertheless, exposure to male-soiled bedding elicits an increase in dopamine efflux in the nucleus accumbens shell and core, measured by microdialysis. Infusion of the opioid antagonist naltrexone in the accumbens core does not significantly affect dopamine efflux during exposure to male chemosignals, although it enhances dopamine levels 40 min after withdrawal of the stimuli. By contrast, infusion of the glutamate antagonist kynurenic acid in the accumbens shell inhibits the release of dopamine and reduces the time that females spend investigating male-soiled bedding. These data are in agreement with previous reports in male rats showing that exposure to opposite-sex odors elicits dopamine release in the accumbens, and with data in female mice showing that the behavioral preference for male chemosignals is not affected by opioidergic antagonists. We hypothesize that glutamatergic projections from the amygdala into the accumbens might be important to modulate the neurochemical and behavioral responses elicited by sexual chemosignals in rats.

Acetaldehyde sequestration by D-penicillamine prevents ethanol relapse-like drinking in rats: evidence from an operant self-administration paradigm.

RATIONALE: Previous experiments in our laboratory have shown that D-penicillamine (DP) (acetaldehyde sequestering agent) is able to block the increase in ethanol consumption observed after a period of imposed deprivation (the so-called alcohol deprivation effect (ADE)), using a non-operant paradigm in Wistar rats. OBJECTIVES: This study is aimed at investigating the robustness and reproducibility of our previous data using an operant paradigm, which is considered to be a valid and reliable model of human drug consumption, and the ADE, probably the most often used measure of ethanol relapse-drinking behaviour in rats. METHODS: Male Wistar rats with a limited (30-min sessions), intermittent and extended background of ethanol operant self-administration were used. In order to evaluate the efficacy of several DP doses (6.25, 12.5 and 25 mg/kg i.p.) in preventing alcohol relapse, we set up a protocol based on the ADE. In a separate experiment, the effect of DP on spontaneous motor activity of rats was also tested. RESULTS: A significant ADE was observed in animals treated with saline. DP treatment blocked the increase in ethanol responses following the imposed abstinence period. The higher dose suppressed the ADE and provoked a significant reduction in ethanol consumption with respect to the baseline conditions. Basal motor activity was not altered after DP treatment. CONCLUSION: Our positive results with DP, using two different paradigms that evaluate relapse of ethanol drinking, will help to increase the positive predictive value of pre-clinical experiments and offer a solid base to inspire human studies with DP.

Dual motor responses elicited by ethanol in the posterior VTA: Consequences of the blockade of µ-opioid receptors.

A recent hypothesis, based on electrophysiological and behavioural findings, suggests that ethanol simultaneously exerts opposed effects on the activity of dopamine (DA) neurons in the ventral tegmental area (VTA) through two parallel mechanisms, one promoting and the other reducing the GABA release onto VTA DA neurons. In this sense, the activating effects are mediated by salsolinol, a metabolite of ethanol, acting on the µ-opioid receptors (MORs) located in VTA GABA neurons. The inhibitory effects are, however, triggered by the non-metabolized fraction of ethanol which would cause the GABAA receptors-mediated inhibition of VTA DA neurons. Since both trends tend to offset each other, only the use of appropriate pharmacological tools allows analysis of this phenomenon in depth. Herein, we present new behavioural findings supporting this hypothesis. Motor activity was evaluated in rats after intra-VTA administration of ethanol 35 nmol, an apparently ineffective dose, 24 h after the irreversible blockade of MORs in the VTA with ß-FNA. Our results showed that this pre-treatment turned the initially ineffective ethanol dose into a depressant one, confirming that the activating effect of ethanol can be selectively suppressed without affecting the depressant effects mediated by the non-biotransformed fraction of ethanol.

Disposition of d-penicillamine, a promising drug for preventing alcohol-relapse. Influence of dose, chronic alcohol consumption and age: studies in rats.

Pharmacokinetic studies concerning d-penicillamine (an acetaldehyde sequestering agent) are scarce and have not evaluated the influence of chronic ethanol consumption and age on its disposition. Since recent preclinical studies propose d-penicillamine as a promising treatment for alcohol relapse, the main aim of the present work was to evaluate the influence of these two factors on d-penicillamine disposition in order to guide future clinical studies on the anti-relapse efficacy of this drug in alcoholism. Additionally, the effect of the administered dose was also evaluated. To this end, three studies were carried out. Study 1 assessed the influence of dose on d-penicillamine disposition, whereas studies 2 and 3 evaluated, respectively, the influence of chronic alcohol consumption and age. Rapid intravenous administrations of 2, 10 and 30 mg/kg of d-penicillamine were performed using young or adult ethanol-naïve rats or adult ethanol-experienced (subjected to a long-term ethanol self-administration protocol) rats. Pharmacokinetic parameters were derived from the biexponential model. Statistical analysis of CL, normalized AUC0 (∞) , V1 and k10 revealed that disposition, in the range plasma concentrations assayed, is non-linear both in young ethanol-naïve and in adult ethanol-experienced rats. Notably, no significant changes in t1/2 were detected. Chronic ethanol consumption significantly reduced CL values by 35% without affecting t1/2 . d-Penicillamine disposition was equivalent in young and adult animals. In conclusion, although DP pharmacokinetics is non-linear, the lack of significant alterations of the t1/2 would potentially simplify the clinical use of this drug. Chronic consumption of ethanol also alters d-penicillamine disposition but, again, does not modify t1/2.

Improved effect of the combination naltrexone/D-penicillamine in the prevention of alcohol relapse-like drinking in rats.

Opioid antagonists are licensed drugs for treating alcohol use disorders; nonetheless, clinical studies have evidenced their limited effectiveness. Preclinical findings indicate that opioid receptor (OR) antagonists, such as naltrexone (NTX), reduce the alcohol deprivation effect (ADE). However, a detailed analysis of published data shows the existence of a delayed increase in ethanol consumption after continuous OR blockade, a phenomenon originally called as 'delayed ADE'. We have recently reported that D-penicillamine (DP) is able to prevent ADE through a mechanism dependent on the inactivation of acetaldehyde, the main metabolite of ethanol. Hypothetically, OR activation would be triggered by acetaldehyde after ethanol consumption. Hence, we conjecture that the combination of NTX and DP, due to their distinct but complementary mechanisms to impede OR activation, may be more efficacious in the prevention of the ADE and, specifically, the 'delayed ADE'. Herein, we compare the effects of the combination NTX/DP (NTX: 2×5 mg/kg SC injection daily/DP: SC infusion (0.25 mg/h)) versus NTX on the ADE in long-term ethanol-experienced rats. As expected, NTX-treated animals displayed a delayed ADE. However, NTX/DP treatment prevented this delayed effect. Our present data indicate that this combination therapy shows an adequate anti-relapse preclinical efficacy being able to overcome the preclinical limitations of NTX alone.

Opposite motor responses elicited by ethanol in the posterior VTA: the role of acetaldehyde and the non-metabolized fraction of ethanol.

Recent electrophysiological evidence suggests that ethanol simultaneously exerts opposite effects on the activity of dopamine (DA) neurons in the ventral tegmental area (VTA) through two parallel mechanisms, one promoting and the other reducing the GABA release onto VTA DA neurons. Here we explore the possible behavioural implications of these findings by investigating the role displayed by acetaldehyde (the main metabolite of ethanol) and the non-metabolized fraction of ethanol in motor activity of rats. We analyse the appearance of motor activation or depression after intra-VTA administration of ethanol in rats subjected to different pharmacological pre-treatments designed to preferentially test either the effects of acetaldehyde or the non-metabolized ethanol. Motor activity was evaluated after intra-VTA administration of 35 nmol of ethanol, an apparently ineffective dose that does not modify the motor activity of animals. Pharmacological pre-treatments were used in order to either increase (cyanamide, 10 mg/kg, ip) or decrease (D-penicillamine, 50 mg/kg, ip and sodium azide, 7 mg/kg, ip) acetaldehyde levels in the VTA. Pre-treatments aimed to augment acetaldehyde, increased motor activity of rats. Otherwise, pre-treatments intended to decrease local acetaldehyde levels evoked significant reductions in motor activity that were prevented by the local blockade (bicuculline, 17.5 pmol) of the GABAA receptors. Our findings suggest that the brain-generated acetaldehyde is involved in the stimulant effects of ethanol, whereas the non-biotransformed fraction of ethanol, acting through the GABAA receptors, would account for the depressant effects. The present behavioural findings suggest that ethanol dually modulates the activity of DA neurons.

Efficacy of D-penicillamine, a sequestering acetaldehyde agent, in the prevention of alcohol relapse-like drinking in rats.

RATIONALE: Nowadays, very few approved anti-relapse treatments for alcoholism exist, and their overall efficacy can be considered moderate. An exciting rationale drug development opportunity for the treatment of chronic alcoholism is the use of acetaldehyde sequestering agents. Although these compounds are able to attenuate or prevent most of the behavioral and neurochemical effects of ethanol, the efficacy of acetaldehyde sequestration, by using agents such as D-penicillamine (DP), in relapse prevention has not been studied yet. OBJECTIVES: The aim of this study was to analyze the effects of DP treatment on the alcohol deprivation effect (ADE) in long-term ethanol-experienced rats as a model of relapse behavior and measure drug plasma and brain levels during treatment. METHODS: Rats were subcutaneously implanted with mini-osmotic pumps delivering 0, 0.25, or 1 mg/h of DP during 1 week. The efficacy to prevent ADE was determined. DP plasma and brain levels achieved during its subcutaneous administration were measured. In a second experiment, animals received bilateral infusions of 0 or 1.5 µg/h of DP directly into pVTA, and the appearance of ADE was evaluated. RESULTS: One milligram per hour, but not 0.25 mg/h, DP infusion prevented ADE and reduced the total ethanol preference in animals. DP plasma concentrations associated with ADE suppression were around 3-4 µg/ml, and brain DP levels in these conditions were about 2-3 % of those found in plasma. Intra-pVTA DP administration also suppressed ADE. CONCLUSION: DP is able to prevent alcohol-relapse-like drinking in rats suggesting that this drug may be a useful new tool in the management of relapse in alcohol-dependent patients.

Salsolinol stimulates dopamine neurons in slices of posterior ventral tegmental area indirectly by activating µ-opioid receptors.

Previous studies in vivo have shown that salsolinol, the condensation product of acetaldehyde and dopamine, has properties that may contribute to alcohol abuse. Although opioid receptors, especially the µ-opioid receptors (MORs), may be involved, the cellular mechanisms mediating the effects of salsolinol have not been fully explored. In the current study, we used whole-cell patch-clamp recordings to examine the effects of salsolinol on dopamine neurons of the ventral tegmental area (VTA) in acute brain slices from Sprague-Dawley rats. Salsolinol (0.01-1 µM) dose-dependently and reversibly increased the ongoing firing of dopamine neurons; this effect was blocked by naltrexone, an antagonist of MORs, and gabazine, an antagonist of GABA(A) receptors. We further showed that salsolinol reduced the frequency without altering the amplitude of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic currents in dopamine neurons. The salsolinol-induced reduction was blocked by both naltrexone and [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, an agonist of MORs. Thus, salsolinol excites VTA-dopamine neurons indirectly by activating MORs, which inhibit GABA neurons in the VTA. This form of disinhibition seems to be a novel mechanism underlying the effects of salsolinol.

Revisiting the controversial role of salsolinol in the neurobiological effects of ethanol: old and new vistas.

The possible involvement of salsolinol (Sal), an endogenous condensation product of ACD (the first metabolite of ethanol) and dopamine, in the neurochemical basis underlying ethanol action has been repeatedly suggested although it has not been unequivocally established, still being a controversial matter of debate. The main goal of this review is to evaluate the presumed contribution of Sal to ethanol effects summarizing the reported data since the discovery in the 1970s of Sal formation in vitro during ethanol metabolism until the more recent studies characterizing its behavioral and neurochemical effects. Towards this end, we first analyze the production and detection of Sal, in different brain areas, in basal conditions and after alcohol consumption, highlighting its presence in regions especially relevant in regulating ethanol-drinking behaviour and the importance of the newly developed methods to differentiate both enantiomers of Sal which could help to explain some previous negative findings. Afterwards, we review the behavioral and neurochemical studies. Finally, we present and discuss the previous and current enunciated mechanisms of action of Sal in the CNS.