A non-hallucinogenic psychedelic analogue with therapeutic potential.

The psychedelic alkaloid ibogaine has anti-addictive properties in both humans and animals1. Unlike most medications for the treatment of substance use disorders, anecdotal reports suggest that ibogaine has the potential to treat addiction to various substances, including opiates, alcohol and psychostimulants. The effects of ibogaine-like those of other psychedelic compounds-are long-lasting2, which has been attributed to its ability to modify addiction-related neural circuitry through the activation of neurotrophic factor signalling3,4. However, several safety concerns have hindered the clinical development of ibogaine, including its toxicity, hallucinogenic potential and tendency to induce cardiac arrhythmias. Here we apply the principles of function-oriented synthesis to identify the key structural elements of the potential therapeutic pharmacophore of ibogaine, and we use this information to engineer tabernanthalog-a water-soluble, non-hallucinogenic, non-toxic analogue of ibogaine that can be prepared in a single step. In rodents, tabernanthalog was found to promote structural neural plasticity, reduce alcohol- and heroin-seeking behaviour, and produce antidepressant-like effects. This work demonstrates that, through careful chemical design, it is possible to modify a psychedelic compound to produce a safer, non-hallucinogenic variant that has therapeutic potential.

The Small G-Protein Rac1 in the Dorsomedial Striatum Promotes Alcohol-Dependent Structural Plasticity and Goal-Directed Learning in Mice.

The small G-protein Ras-related C3 botulinum toxin substrate 1 (Rac1) promotes the formation of filamentous actin (F-actin). Actin is a major component of dendritic spines, and we previously found that alcohol alters actin composition and dendritic spine structure in the nucleus accumbens (NAc) and the dorsomedial striatum (DMS). To examine if Rac1 contributes to these alcohol-mediated adaptations, we measured the level of GTP-bound active Rac1 in the striatum of mice following 7 weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS of male mice. In contrast, Rac1 is not activated by alcohol in the NAc and DLS of male mice or in the DMS of female mice. Similarly, closely related small G-proteins are not activated by alcohol in the DMS, and Rac1 activity is not increased in the DMS by moderate alcohol or natural reward. To determine the consequences of alcohol-dependent Rac1 activation in the DMS of male mice, we inhibited endogenous Rac1 by infecting the DMS of mice with an adeno-associated virus (AAV) expressing a dominant negative form of the small G-protein (Rac1-DN). We found that overexpression of AAV-Rac1-DN in the DMS inhibits alcohol-mediated Rac1 signaling and attenuates alcohol-mediated F-actin polymerization, which corresponded with a decrease in dendritic arborization and spine maturation. Finally, we provide evidence to suggest that Rac1 in the DMS plays a role in alcohol-associated goal-directed learning. Together, our data suggest that Rac1 in the DMS plays an important role in alcohol-dependent structural plasticity and aberrant learning.

Molecular mechanisms underlying alcohol-drinking behaviours.

The main characteristic of alcohol use disorder is the consumption of large quantities of alcohol despite the negative consequences. The transition from the moderate use of alcohol to excessive, uncontrolled alcohol consumption results from neuroadaptations that cause aberrant motivational learning and memory processes. Here, we examine studies that have combined molecular and behavioural approaches in rodents to elucidate the molecular mechanisms that keep the social intake of alcohol in check, which we term 'stop pathways', and the neuroadaptations that underlie the transition from moderate to uncontrolled, excessive alcohol intake, which we term 'go pathways'. We also discuss post-transcriptional, genetic and epigenetic alterations that underlie both types of pathways.

Differential correlation of serum BDNF and microRNA content in rats with rapid or late onset of heavy alcohol use.

Heavy alcohol use reduces the levels of the brain-derived neurotrophic factor (BDNF) in the prefrontal cortex of rodents through the upregulation of microRNAs (miRs) targeting BDNF mRNA. In humans, an inverse correlation exists between circulating blood levels of BDNF and the severity of psychiatric disorders including alcohol abuse. Here, we set out to determine whether a history of heavy alcohol use produces comparable alterations in the blood of rats. We used an intermittent access to 20% alcohol using the two-bottle choice paradigm (IA20%2BC) and measured circulating levels of BDNF protein and miRs targeting BDNF in the serum of Long-Evans rats before and after 8 weeks of excessive alcohol intake. We observed that the drinking profile of heavy alcohol users is not unified, whereas 70% of the rats gradually escalate their alcohol intake (late onset), and 30% of alcohol users exhibit a very rapid onset of drinking (rapid onset). We found that serum BDNF levels are negatively correlated with alcohol intake in both rapid onset and late onset rats. In contrast, increased expression of the miRs targeting BDNF, miR30a-5p, miR-195-5p, miR191-5p and miR206-3p, was detected only in the rapid onset rats. Finally, we report that the alcohol-dependent molecular changes are not due to alterations in platelet number. Together, these data suggest that rats exhibit both late and rapid onset of alcohol intake. We further show that heavy alcohol use produces comparable changes in BDNF protein levels in both groups. However, circulating microRNAs are responsive to alcohol only in the rapid onset rats.

The Fyn kinase inhibitor, AZD0530, suppresses mouse alcohol self-administration and seeking.

Fyn is a member of the Src family of protein tyrosine kinases (PTKs) that plays an important role not only in normal synaptic functions but also in brain pathologies including alcohol use disorder. We previously reported that repeated cycles of binge drinking and withdrawal activate Fyn in the dorsomedial striatum (DMS) of rodents, and that Fyn signaling in the DMS contributes to rat alcohol intake and relapse. Here, we used AZD0530, a CNS penetrable inhibitor of Src PTKs developed for the treatment of Alzheimer disease and cancer and tested its efficacy to suppress alcohol-dependent molecular and behavioral effects. We show that systemic administration of AZD0530 prevents alcohol-induced Fyn activation and GluN2B phosphorylation in the DMS of mice. We further report that a single dose of AZD0530 reduces alcohol operant self-administration and promotes extinction of alcohol self-administration without altering basal and dopamine D1 receptor-dependent locomotion. Together, our findings suggest that AZD0530, through its inhibitory actions on Fyn kinase, dampens alcohol seeking and drinking.

GDNF and alcohol use disorder.

Glial cell line-derived neurotrophic factor (GDNF) has been extensively studied for its role in the development and maintenance of the midbrain dopaminergic system, although evidence suggests that GDNF also plays a role in drug and alcohol addiction. This review focuses on the unique actions of GDNF in the mechanisms that prevent the transition from recreational alcohol use to abuse. Specifically, we describe studies in rodents suggesting that alcohol acutely increases GDNF expression in the ventral tegmental area, which enables the activation of the mitogen-activated protein kinase signaling pathway and the gating of alcohol intake. We further provide evidence to suggest that GDNF acts in the ventral tegmental area via both nongenomic and genomic mechanisms to suppress alcohol consumption. In addition, we describe findings indicating that when this endogenous protective pathway becomes dysregulated, alcohol intake levels escalate. Finally, we describe the potential use of GDNF inducers as a novel therapeutic approach to treat alcohol use disorder.

Mammalian target of rapamycin complex 1 and its downstream effector collapsin response mediator protein-2 drive reinstatement of alcohol reward seeking.

Alcohol use disorder is a chronic relapsing disease. Maintaining abstinence represents a major challenge for alcohol-dependent patients. Yet the molecular underpinnings of alcohol relapse remain poorly understood. In the present study, we investigated the potential role of the mammalian target of rapamycin complex 1 (mTORC1) in relapse to alcohol-seeking behavior by using the reinstatement of a previously extinguished alcohol conditioned place preference (CPP) response as a surrogate relapse paradigm. We found that mTORC1 is activated in the nucleus accumbens shell following alcohol priming-induced reinstatement of alcohol place preference. We further report that the selective mTORC1 inhibitor, rapamycin, abolishes reinstatement of alcohol place preference. Activation of mTORC1 initiates the translation of synaptic proteins, and we observed that reinstatement of alcohol CPP is associated with increased protein levels of one of mTORC1's downstream targets, collapsin response mediator protein-2 (CRMP2), in the nucleus accumbens. Importantly, the level of mTORC1 activation and CRMP2 expression positively correlate with the CPP score during reinstatement. Finally, we found that systemic administration of the CRMP2 inhibitor, lacosamide, attenuates alcohol priming-induced reinstatement of CPP. Together, our results reveal that mTORC1 and its downstream target, CRMP2, contribute to mechanisms underlying reinstatement of alcohol reward seeking. Our results could have important implications for the treatment of relapse to alcohol use and position the Food and Drug Administration approved drugs, rapamycin and lacosamide, for the treatment of alcohol use disorder.

A new generation of mTORC1 inhibitor attenuates alcohol intake and reward in mice.

Alcohol use disorder (AUD) is a chronic condition associated with devastating socioeconomic consequences. Yet, pharmacotherapies to treat behavioral phenotypes such as uncontrolled heavy drinking are limited. Studies in rodents suggest that the mammalian target of rapamycin complex 1 (mTORC1) plays an important role in mechanisms underlying alcohol drinking behaviors as well as alcohol seeking and relapse. These preclinical evidence suggest that mTORC1 may be a therapeutic target for the treatment of AUD. Thus, the aim of the present study was to test the potential use of newly developed mTORC1 inhibitors, RapaLink-1 and MLN0128, in preclinical mouse models of AUD. First, we used the intermittent access to 20 percent alcohol in a two-bottle choice paradigm and tested the efficacy of the drugs to reduce alcohol intake in mice with a history of binge drinking and withdrawal. We found that both inhibitors reduce excessive alcohol intake and preference with RapaLink-1 exhibiting higher efficacy. We further observed that RapaLink-1 attenuates alcohol consumption during the first alcohol-drinking session in naïve mice, and interestingly, the effect was still present 14 days after the initial treatment with the drug. We also found that RapaLink-1 did not alter the consumption of water or saccharin, revealing a specific effect of the inhibitor on alcohol intake. Finally, we report that RapaLink-1 blocks the retrieval but not acquisition of alcohol place preference without affecting locomotion. Together, our findings suggest that RapaLink-1 may be developed as a new medication to treat and prevent the development of AUD.

The First Alcohol Drink Triggers mTORC1-Dependent Synaptic Plasticity in Nucleus Accumbens Dopamine D1 Receptor Neurons.

Early binge-like alcohol drinking may promote the development of hazardous intake. However, the enduring cellular alterations following the first experience with alcohol consumption are not fully understood. We found that the first binge-drinking alcohol session produced enduring enhancement of excitatory synaptic transmission onto dopamine D1 receptor-expressing neurons (D1+ neurons) in the nucleus accumbens (NAc) shell but not the core in mice, which required D1 receptors (D1Rs) and mechanistic target of rapamycin complex 1 (mTORC1). Furthermore, inhibition of mTORC1 activity during the first alcohol drinking session reduced alcohol consumption and preference of a subsequent drinking session. mTORC1 is critically involved in RNA-to-protein translation, and we found that the first alcohol session rapidly activated mTORC1 in NAc shell D1+ neurons and increased synaptic expression of the AMPAR subunit GluA1 and the scaffolding protein Homer. Finally, D1R stimulation alone was sufficient to activate mTORC1 in the NAc to promote mTORC1-dependent translation of the synaptic proteins GluA1 and Homer. Together, our results indicate that the first alcohol drinking session induces synaptic plasticity in NAc D1+ neurons via enhanced mTORC1-dependent translation of proteins involved in excitatory synaptic transmission that in turn drives the reinforcement learning associated with the first alcohol experience. Thus, the alcohol-dependent D1R/mTORC1-mediated increase in synaptic function in the NAc may reflect a neural imprint of alcohol's reinforcing properties, which could promote subsequent alcohol intake. Significance statement: Consuming alcohol for the first time is a learning event that drives further drinking. Here, we identified a mechanism that may underlie the reinforcing learning associated with the initial alcohol experience. We show that the first alcohol experience induces a persistent enhancement of excitatory synaptic transmission on NAc shell D1+ neurons, which is dependent on D1R and mTORC1. We also find that mTORC1 is necessary for the sustained alcohol consumption and preference across the initial drinking sessions. The first alcohol binge activates mTORC1 in NAc D1+ neurons and increases levels of synaptic proteins involved in glutamatergic signaling. Thus, the D1R/mTORC1-dependent plasticity following the first alcohol exposure may be a critical cellular component of reinforcement learning.

The Neurotrophic Factor Receptor p75 in the Rat Dorsolateral Striatum Drives Excessive Alcohol Drinking.

UNLABELLED: Brain-derived neurotrophic factor (BDNF) signaling in the dorsolateral striatum (DLS) keeps alcohol intake in moderation. For example, activation of the BDNF receptor tropomyosin receptor kinase B (TrkB) in the DLS reduces intake in rats that consume moderate amounts of alcohol. Here, we tested whether long-term excessive consumption of alcohol produces neuroadaptations in BDNF signaling in the rat DLS. We found that BDNF was no longer able to gate alcohol self-administration after a history of repeated cycles of binge alcohol drinking and withdrawal. We then elucidated the possible neuroadaptations that could block the ability of BDNF to keep consumption of alcohol in moderation. We report that intermittent access to 20% alcohol in a two-bottle choice paradigm that models excessive alcohol drinking produces a mobilization of DLS p75 neurotrophin receptor (p75NTR), whose activities oppose those of the Trk receptors, including TrkB. These neuroadaptations were not observed in the DLS of rats exposed to continuous access to 10% alcohol or in rats consuming sucrose. Furthermore, short hairpin RNA (shRNA)-mediated knockdown of the p75NTR gene in the DLS, as well as intra-DLS infusion or systemic administration of the p75NTR modulator, LM11A-31, significantly reduced binge drinking of alcohol. Together, our results suggest that excessive alcohol consumption produces a change in BDNF signaling in the DLS, which is mediated by the recruitment of p75NTR. Our data also imply that modulators of p75NTR signaling could be developed as medications for alcohol abuse disorders. SIGNIFICANCE STATEMENT: Neuroadaptations gate or drive excessive, compulsive alcohol drinking. We previously showed that brain-derived neurotrophic factor and its receptor, TrkB, in the dorsolateral striatum (DLS), are part of an endogenous system that keeps alcohol drinking in moderation. Here, we show that a history of excessive alcohol intake produces neuroadaptations in the DLS that preclude BDNF's ability to gate alcohol self-administration in rats by the recruitment of the low-affinity neurotrophin receptor, p75NTR, whose activities opposes those of the Trk receptors. Finally, we show that the administration of the p75NTR modulator, LM11A-31, significantly reduces excessive alcohol intake suggesting that the drug may be developed as a new treatment for alcohol abuse disorders.

Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice.

BACKGROUND: Liver damage is a serious and sometimes fatal consequence of long-term alcohol intake, which progresses from early-stage fatty liver (steatosis) to later-stage steatohepatitis with inflammation and fibrosis/necrosis. However, very little is known about earlier stages of liver disruption that may occur in problem drinkers, those who drink excessively but are not dependent on alcohol. METHODS: We examined how repeated binge-like alcohol drinking in C57BL/6 mice altered liver function, as compared with a single binge-intake session and with repeated moderate alcohol consumption. We measured a number of markers associated with early- and later-stage liver disruption, including liver steatosis, measures of liver cytochrome P4502E1 (CYP2E1) and alcohol dehydrogenase (ADH), alcohol metabolism, expression of cytokine mRNA, accumulation of 4-hydroxynonenal (4-HNE) as an indicator of oxidative stress, and alanine transaminase/aspartate transaminase as a measure of hepatocyte injury. RESULTS: Importantly, repeated binge-like alcohol drinking increased triglyceride levels in the liver and plasma, and increased lipid droplets in the liver, indicators of steatosis. In contrast, a single binge-intake session or repeated moderate alcohol consumption did not alter triglyceride levels. In addition, alcohol exposure can increase rates of alcohol metabolism through CYP2E1 and ADH, which can potentially increase oxidative stress and liver dysfunction. Intermittent, excessive alcohol intake increased liver CYP2E1 mRNA, protein, and activity, as well as ADH mRNA and activity. Furthermore, repeated, binge-like drinking, but not a single binge or moderate drinking, increased alcohol metabolism. Finally, repeated, excessive intake transiently elevated mRNA for the proinflammatory cytokine IL-1B and 4-HNE levels, but did not alter markers of later-stage liver hepatocyte injury. CONCLUSIONS: Together, we provide data suggesting that even relatively limited binge-like alcohol drinking can lead to disruptions in liver function, which might facilitate the transition to more severe forms of liver damage.

Region specific activation of the AKT and mTORC1 pathway in response to excessive alcohol intake in rodents.

We previously reported that the kinase AKT is activated in the nucleus accumbens (NAc) of rodents in response to excessive consumption of alcohol. One of the important downstream targets of AKT is the mammalian Target Of Rapamycin in Complex 1 (mTORC1), which was also activated by alcohol intake. mTORC1 controls dendritic protein translation, and we showed that the mTORC1-dependent translational machinery is activated in the NAc in response to alcohol intake. Importantly, systemic or intra-NAc inhibition of the AKT/mTORC1 pathway attenuated alcohol-drinking behaviors. Here, we mapped the activation patterns of AKT and mTORC1 in corticostriatal regions of rodents consuming large amounts of alcohol. We found that the activation of AKT and mTORC1 in response to cycles of binge drinking of 20 percent alcohol was centered in the NAc shell. Both kinases were not activated in the dorsolateral striatum (DLS); however, AKT, but not mTORC1, was activated in the dorsomedial striatum (DMS) of mice but not rats. Interestingly, excessive intake of alcohol produced a selective activation of the AKT/mTORC1 pathway in the orbitofrontal cortex (OFC), which was not observed in medial prefrontal cortex (mPFC). Furthermore, this signaling pathway was not activated in the NAc shell or OFC of rats consuming moderate amounts of alcohol nor was it activated in rats consuming sucrose. Together, our results suggest that excessive alcohol intake produces a brain region selective activation of the AKT/mTORC1 pathway, which is likely to contribute to NAc shell and OFC-dependent mechanisms that underlie the development and maintenance of alcohol drinking behavior.

Alcohol Elicits Functional and Structural Plasticity Selectively in Dopamine D1 Receptor-Expressing Neurons of the Dorsomedial Striatum.

Addiction is thought to be a maladaptive form of learning and memory caused by drug-evoked aberrant synaptic plasticity. We previously showed that alcohol facilitates synaptic plasticity in the dorsomedial striatum (DMS), a brain region that drives goal-directed behaviors. The majority of DMS cells are medium spiny neurons (MSNs) that express dopamine D1 receptors (D1Rs) or D2 receptors (D2Rs), which drive "Go" or "No-Go" behaviors, respectively. Here, we report that alcohol induces cell type-specific synaptic and structural plasticity in the DMS. Using mice that express a fluorescence marker to visualize D1R or D2R MSNs, we show that repeated cycles of systemic administration of alcohol or alcohol consumption induces a long-lasting increase in AMPAR activity specifically in DMS D1R but not in D2R MSNs. Importantly, we report that alcohol consumption increases the complexity of dendritic branching and the density of mature mushroom-shaped spines selectively in DMS D1R MSNs. Finally, we found that blockade of D1R but not D2R activity in the DMS attenuates alcohol consumption. Together, these data suggest that alcohol intake produces profound functional and structural plasticity events in a subpopulation of neurons in the DMS that control reinforcement-related learning. SIGNIFICANCE STATEMENT: Alcohol addiction is considered maladaptive learning and memory processes. Here we unraveled a long-lasting cellular mechanism that may contribute to the memory of alcohol-seeking behaviors. Specifically, we found that alcohol consumption produces a long-lasting enhancement of channel activity and persistent alterations of neuronal morphology in a part of the brain (DMS) that controls alcohol-drinking behaviors. Furthermore, we show that these alterations occur only in a subpopulation of neurons that positively control reward and reinforcement of drugs of abuse. Finally, we report that blocking the activity of this neuronal population reduces alcohol intake. As such synaptic and structural changes are the cellular hallmarks of learning and memory, and these neuroadaptations may drive the development of pathological heavy alcohol consumption.

Glial cell line-derived neurotrophic factor (GDNF) is an endogenous protector in the mesolimbic system against excessive alcohol consumption and relapse.

Moderate social consumption of alcohol is common; however, only a small percentage of individuals transit from social to excessive, uncontrolled alcohol drinking. This suggests the existence of protective mechanisms that prevent the development of alcohol addiction. Here, we tested the hypothesis that the glial cell line-derived neurotrophic factor (GDNF) in the mesolimbic system [e.g. the nucleus accumbens (Acb) and ventral tegmental area (VTA)] is part of such a mechanism. We found that GDNF knockdown, by infecting rat Acb neurons with a small hairpin RNA (shRNA) targeting the GDNF gene, produced a rapid escalation to excessive alcohol consumption and enhanced relapse to alcohol drinking. Conversely, viral-mediated overexpression of the growth factor in the mesolimbic system blocked the escalation from moderate to excessive alcohol drinking. To access the mechanism underlying GDNF's actions, we measured the firing rate of dopaminergic (DAergic) neurons in the VTA after a history of excessive alcohol intake with or without elevating GDNF levels. We found that the spontaneous firing rate of DAergic neurons in the VTA was reduced during alcohol withdrawal and that GDNF reversed this alcohol-induced DA deficiency. Together, our results suggest that endogenous GDNF in the mesolimbic system controls the transition from moderate to excessive alcohol drinking and relapse via reversal of alcohol-dependent neuro-adaptations in DAergic VTA neurons.

Protein tyrosine phosphatase α in the dorsomedial striatum promotes excessive ethanol-drinking behaviors.

We previously found that excessive ethanol drinking activates Fyn in the dorsomedial striatum (DMS) (Wang et al., 2010; Gibb et al., 2011). Ethanol-mediated Fyn activation in the DMS leads to the phosphorylation of the GluN2B subunit of the NMDA receptor, to the enhancement of the channel's activity, and to the development and/or maintenance of ethanol drinking behaviors (Wang et al., 2007, 2010). Protein tyrosine phosphatase α (PTPα) is essential for Fyn kinase activation (Bhandari et al., 1998), and we showed that ethanol-mediated Fyn activation is facilitated by the recruitment of PTPα to synaptic membranes, the compartment where Fyn resides (Gibb et al., 2011). Here we tested the hypothesis that PTPα in the DMS is part of the Fyn/GluN2B pathway and is thus a major contributor to the neuroadaptations underlying excessive ethanol intake behaviors. We found that RNA interference (RNAi)-mediated PTPα knockdown in the DMS reduces excessive ethanol intake and preference in rodents. Importantly, no alterations in water, saccharine/sucrose, or quinine intake were observed. Furthermore, downregulation of PTPα in the DMS of mice significantly reduces ethanol-mediated Fyn activation, GluN2B phosphorylation, and ethanol withdrawal-induced long-term facilitation of NMDAR activity without altering the intrinsic features of DMS neurons. Together, these results position PTPα upstream of Fyn within the DMS and demonstrate the important contribution of the phosphatase to the maladaptive synaptic changes that lead to excessive ethanol intake.

mTOR complex 1: a key player in neuroadaptations induced by drugs of abuse.

The mammalian (or mechanistic) target of rapamycin (mTOR) complex 1 (mTORC1) is a serine and threonine kinase that regulates cell growth, survival, and proliferation. mTORC1 is a master controller of the translation of a subset of mRNAs. In the central nervous system mTORC1 plays a crucial role in mechanisms underlying learning and memory by controlling synaptic protein synthesis. Here, we review recent evidence suggesting that the mTORC1 signaling pathway promotes neuroadaptations following exposure to a diverse group of drugs of abuse including stimulants, cannabinoids, opiates, and alcohol. We further describe potential molecular mechanisms by which drug-induced mTORC1 activation may alter brain functions. Finally, we propose that mTORC1 is a focal point shared by drugs of abuse to mediate drug-related behaviors such as reward seeking and excessive drug intake, and offer future directions to decipher the contribution of the kinase to mechanisms underlying addiction. Recent studies suggesting that exposure to diverse classes of drugs of abuse as well as exposure to drug-associated memories lead to mTORC1 kinase activation in the limbic system. In turn, mTORC1 controls the onset and the maintenance of pathological neuroadaptions that underlie several features of drug addiction such as drug seeking and relapse. Therefore, we propose that targeting mTORC1 and its effectors is a promising strategy to treat drug disorders.

Inhibition of striatal-enriched tyrosine phosphatase 61 in the dorsomedial striatum is sufficient to increased ethanol consumption.

The STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61 ) inhibits the activity of the tyrosine kinase Fyn and dephosphorylates the GluN2B subunit of the NMDA receptor, whereas the protein kinase A phosphorylation of STEP61 inhibits the activity of the phosphatase (Pharmacol. Rev., 64, , p. 65). Previously, we found that ethanol activates Fyn in the dorsomedial striatum (DMS) leading to GluN2B phosphorylation, which, in turn, underlies the development of ethanol intake (J. Neurosci., 30, , p. 10187). Here, we tested the hypothesis that inhibition of STEP61 by ethanol is upstream of Fyn/GluN2B. We show that exposure of mice to ethanol increased STEP61 phosphorylation in the DMS, which was maintained after withdrawal and was not observed in other striatal regions. Specific knockdown of STEP61 in the DMS of mice enhanced ethanol-mediated Fyn activation and GluN2B phosphorylation, and increased ethanol intake without altering the level of water, saccharine, quinine consumption or spontaneous locomotor activity. Together, our data suggest that blockade of STEP61 activity in response to ethanol is sufficient for the activation of the Fyn/GluN2B pathway in the DMS. Being upstream of Fyn and GluN2B, inactive STEP61 in the DMS primes the induction of ethanol intake. We show that ethanol-mediated inhibition of STEP61 in the DMS leads to Fyn activation and GluN2B phosphorylation. (a) Under basal conditions, active STEP61 inhibits Fyn activity and dephosphorylates GluN2B. (b) Ethanol leads to the phosphorylation of STEP61 on a specific inhibitory site. The inhibition of STEP61 activity contributes to the activation of Fyn in response to ethanol, which, in turn, phosphorylates GluN2B. These molecular adaptations in the DMS promote ethanol drinking.

GDNF is a novel ethanol-responsive gene in the VTA: implications for the development and persistence of excessive drinking.

Glial cell line-derived neurotrophic factor (GDNF) is a potent inhibitor of ethanol consumption and relapse, and GDNF heterozygous knockout mice display increased reward sensitivity to ethanol and consume more ethanol after a period of abstinence than their wild-type littermates. Here, we tested whether ethanol alters GDNF expression in the ventral tegmental area (VTA; GDNF's site of action) and/or the nucleus accumbens (NAc; the main source of GDNF), and if so, determine the role of the endogenous growth factor in the regulation of ethanol consumption. Systemic administration of ethanol increased GDNF expression and protein levels in the VTA, but not the NAc. Additionally, GDNF levels were elevated after an ethanol-drinking session in rats that consumed ethanol in the intermittent-access two-bottle choice procedure for 1 week, but not 7 weeks. Deprivation following 7 weeks of excessive ethanol intake reduced GDNF levels, while a short ethanol binge drinking period following deprivation upregulated GDNF expression. Importantly, knockdown of GDNF within the VTA using adenovirus expressing short hairpin RNA facilitated the escalation of ethanol drinking by ethanol-naïve rats, but not by rats with a history of excessive ethanol consumption. These results suggest that during initial ethanol-drinking experiences, GDNF in the VTA is increased and protects against the development of excessive ethanol intake. However, the growth factor's protective response to ethanol breaks down after protracted excessive ethanol intake and withdrawal, resulting in persistent, excessive ethanol consumption.

Extrasynaptic delta-containing GABAA receptors in the nucleus accumbens dorsomedial shell contribute to alcohol intake.

Recent findings suggest that extrasynaptic δ-subunit-containing GABA(A) receptors are sensitive to low-to-moderate concentrations of alcohol, raising the possibility that these receptors mediate the reinforcing effects of alcohol after consumption of one or a few drinks. We used the technique of viral-mediated RNAi to reduce expression of the GABA(A) receptor δ-subunit in adult rats in localized regions of the nucleus accumbens (NAc) to test the hypothesis that δ-subunit-containing GABA(A) receptors in the NAc are necessary for oral alcohol consumption. We found that knockdown of the δ-subunit in the medial shell region of the NAc, but not in the ventral or lateral shell or in the core, reduced alcohol intake. In contrast, δ-subunit knockdown in the medial shell did not affect intake of a 2% sucrose solution, suggesting that the effects of GABA(A) receptor δ-subunit reduction are specific to alcohol. These results provide strong evidence that extrasynaptic δ-subunit-containing GABA(A) receptors in the medial shell of the NAc are critical for the reinforcing effects of oral ethanol.

Sex Differences In The Interaction Between Alcohol And mTORC1.

The kinase mechanistic target of rapamycin complex 1 (mTORC1) plays an essential role in learning and memory by promoting mRNA to protein translation of a subset of synaptic proteins at dendrites. We generated a large body of data in male rodents indicating that mTORC1 is critically involved in mechanisms that promote numerous adverse behaviors associated with alcohol use disorder (AUD) including heavy alcohol use. For example, we found that mTORC1 is activated in the nucleus accumbens (NAc) and orbitofrontal cortex (OFC) of male mice and rats that were subjected to 7 weeks of intermittent access to 20% alcohol two-bottle choice (IA20%2BC). We further showed that systemic or intra-NAc administration of the selective mTORC1 inhibitor, rapamycin, decreases alcohol seeking and drinking, whereas intra-OFC administration of rapamycin reduces alcohol seeking and habit in male rats. This study aimed to assess mTORC1 activation in these corticostriatal regions of female mice and to determine whether the selective mTORC1 inhibitor, rapamycin, can be used to reduce heavy alcohol use in female mice. We found that mTORC1 is not activated by 7 weeks of intermittent 20% alcohol binge drinking and withdrawal in the NAc and OFC. Like in males, mTORC1 signaling was not activated by chronic alcohol intake and withdrawal in the medial prefrontal cortex (mPFC) of female mice. Interestingly, Pearson correlation comparisons revealed that the basal level of mTORC1 activation between the two prefrontal regions, OFC and mPFC were correlated and that the drinking profile predicts the level of mTORC1 activation in the mPFC after 4-hour binge drinking. Finally, we report that administration of rapamycin does not attenuate heavy alcohol drinking in female animals. Together, our results suggest a sex-dependent contribution of mTORC1 to the neuroadaptation that drives alcohol use and abuse.