Ethanol-related Behaviors Research Articles

Serotonin transporter knockdown relieves depression-like behavior and ethanol-induced CPP in mice after chronic social defeat stress

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

Forced Abstinence from Volitional Ethanol Intake Drives a Vulnerable Period of Hyperexcitability in BNST-Projecting Insular Cortex Neurons.

The insular cortex (IC) integrates sensory and interoceptive cues to inform downstream circuitry executing adaptive behavioral responses. The IC communicates with areas involved canonically in stress and motivation. IC projections govern stress and ethanol recruitment of bed nucleus of the stria terminalis (BNST) activity necessary for the emergence of negative affective behaviors during alcohol abstinence. Here, we assess the impact of the chronic drinking forced abstinence (CDFA) volitional home cage ethanol intake paradigm on synaptic and excitable properties of IC neurons that project to the BNST (IC→BNST). Using whole-cell patch-clamp electrophysiology, we investigated IC→BNST circuitry 24 h or 2 weeks following forced abstinence (FA) in female C57BL6/J mice. We find that IC→BNST cells are transiently more excitable following acute ethanol withdrawal. In contrast, in vivo ethanol exposure via intraperitoneal injection, ex vivo via ethanol wash, and acute FA from a natural reward (sucrose) all failed to alter excitability. In situ hybridization studies revealed that at 24 h post FA BK channel mRNA expression is reduced in IC. Further, pharmacological inhibition of BK channels mimicked the 24 h FA phenotype, while BK activation was able to decrease AP firing in control and 24 h FA subjects. All together these data suggest a novel mechanism of homeostatic plasticity that occurs in the IC→BNST circuitry following chronic drinking.

Gestational environmental enrichment prevents chronic social stress induced anxiety- and ethanol-related behaviors in offspring

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

Anxiety and ethanol consumption in socially defeated mice; effect of hippocampal serotonin transporter knockdown

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

Chronic knockdown of the tetraspanin gene CD81 in the mouse nucleus accumbens modulates anxiety and ethanol-related behaviors

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

Contribution of GlyR α3 Subunits to the Sensitivity and Effect of Ethanol in the Nucleus Accumbens.

The glycine receptor (GlyR), a ligand-gated ion channel, is critical for inhibitory neurotransmission in brainstem, spinal cord, and in supraspinal regions. Recent data from several laboratories have shown that GlyRs are expressed in the brain reward circuitry and that α1 and α2 are the principal subunits expressed in the nucleus accumbens (nAc). In the present study, we studied the sensitivity to ethanol of homomeric and heteromeric α3 GlyR subunits in HEK293 cells and dissociated neurons from the nAc. Finally, we explored ethanol-related behaviors in a Glra3 knockout mouse (Glra3–/–). Studies in HEK293 cells showed that while homomeric α3 GlyR subunits were insensitive to ethanol, heteromeric α3β GlyR subunits showed higher sensitivity to ethanol. Additionally, using electrophysiological recordings in dissociated accumbal neurons, we found that the glycine current density increased in Glra3–/– mice and the GlyRs were less affected by ethanol and picrotoxin. We also examined the effect of ethanol on sedation and drinking behavior in Glra3–/– mice and found that the duration in the loss of righting reflex (LORR) was unchanged compared to wild-type (WT) mice. On the other hand, using the drinking in the dark (DID) paradigm, we found that Glra3–/– mice have a larger ethanol consumption compared to WT mice, and that this was already high during the first days of exposure to ethanol. Our results support the conclusion that heteromeric α3β, but not homomeric α3, GlyRs are potentiated by ethanol. Also, the increase in GlyR and GABAAR mediated current densities in accumbal neurons in the KO mice support the presence of compensatory changes to α3 knock out. The increase in ethanol drinking in the Glra3–/– mice might be associated to the reduction in β and compensatory changes in other subunits in the receptor arrangement.

Drosophila Corazonin Neurons as a Hub for Regulating Growth, Stress Responses, Ethanol-Related Behaviors, Copulation Persistence and Sexually Dimorphic Reward Pathways.

The neuronal mechanisms by which complex behaviors are coordinated and timed often involve neuropeptidergic regulation of stress and reward pathways. Recent studies of the neuropeptide Corazonin (Crz), a homolog of the mammalian Gonadotrophin Releasing Hormone (GnRH), have suggested its crucial role in the regulation of growth, internal states and behavioral decision making. We focus this review on Crz neurons with the goal to (1) highlight the diverse roles of Crz neuron function, including mechanisms that may be independent of the Crz peptide, (2) emphasize current gaps in knowledge about Crz neuron functions, and (3) propose exciting ideas of novel research directions involving the use of Crz neurons. We describe the different developmental fates of distinct subsets of Crz neurons, including recent findings elucidating the molecular regulation of apoptosis. Crz regulates systemic growth, food intake, stress responses and homeostasis by interacting with the short Neuropeptide F (sNPF) and the steroid hormone ecdysone. Additionally, activation of Crz neurons is shown to be pleasurable by interacting with the Neuropeptide F (NPF) and regulates reward processes such as ejaculation and ethanol-related behaviors in a sexually dimorphic manner. Crz neurons are proposed to be a motivational switch regulating copulation duration using a CaMKII-dependent mechanism described as the first neuronal interval timer lasting longer than a few seconds. Lastly, we propose ideas to use Crz neuron-induced ejaculation to study the effects of fictive mating and sex addiction in flies, as well as to elucidate dimorphic molecular mechanisms underlying reward behaviors and feeding disorders.

Ethanol-Related Behaviors in Mouse Lines Selectively Bred for Drinking to Intoxication.

Alcohol use disorder (AUD) is a devastating psychiatric disorder that has significant wide-reaching effects on individuals and society. Selectively bred mouse lines are an effective means of exploring the genetic and neuronal mechanisms underlying AUD and such studies are translationally important for identifying treatment options. Here, we report on behavioral characterization of two replicate lines of mice that drink to intoxication, the High Drinking in the Dark (HDID)-1 and -2 mice, which have been selectively bred (20+ generations) for the primary phenotype of reaching high blood alcohol levels (BALs) during the drinking in the dark (DID) task, a binge-like drinking assay. Along with their genetically heterogenous progenitor line, Hs/Npt, we tested these mice on: DID and drinking in the light (DIL); temporal drinking patterns; ethanol sensitivity, through loss of righting reflex (LORR); and operant self-administration, including fixed ratio (FR1), fixed ratio 3:1 (FR3), extinction/reinstatement, and progressive ratio (PR). All mice consumed more ethanol during the dark than the light and both HDID lines consumed more ethanol than Hs/Npt during DIL and DID. In the dark, we found that the HDID lines achieved high blood alcohol levels early into a drinking session, suggesting that they exhibit front loading like drinking behavior in the absence of the chronicity usually required for such behavior. Surprisingly, HDID-1 (female and male) and HDID-2 (male) mice were more sensitive to the intoxicating effects of ethanol during the dark (as determined by LORR), while Hs/Npt (female and male) and HDID-2 (female) mice appeared less sensitive. We observed lower HDID-1 ethanol intake compared to either HDID-2 or Hs/Npt during operant ethanol self-administration. There were no genotype differences for either progressive ratio responding, or cue-induced ethanol reinstatement, though the latter is complicated by a lack of extinguished responding behavior. Taken together, these findings suggest that genes affecting one AUD-related behavior do not necessarily affect other AUD-related behaviors. Moreover, these findings highlight that alcohol-related behaviors can also differ between lines selectively bred for the same phenotype, and even between sexes within those same line.

Lentiviral-mediated up-regulation of let-7d microRNA decreases alcohol intake through down-regulating the dopamine D3 receptor

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

7,8-Dihydroxyflavone Attenuates Alcohol-Related Behavior in Rat Models of Alcohol Consumption via TrkB in the Ventral Tegmental Area.

Alcohol use disorder (AUD) is a ubiquitous substance use disorder in the world, of which neural mechanisms remain unclear. Alcohol consumption induces neuro-adaptations in the dopaminergic system originating from the ventral tegmental area (VTA), an important brain region for the reward function in AUD. Endogenous brain-derived neurotrophic factor (BDNF)-TrkB implicated in the development of neuroplasticity, including long-term potentiation of GABAergic synapses (LTPGABA). We previously found that ethanol blocks LTPGABA in the VTA, either in vivo or in vitro. 7,8-dihydroflavone (7,8-DHF), a BDNF-mimicking small compound, was recently found to penetrate the blood–brain barrier to mimic the biological role of BDNF-TrkB. In this study, we demonstrate that repeated ethanol consumption (including intermittent and continuous ethanol exposure) results in low expression of BDNF in rat VTA. The amount of ethanol intake enhances significantly in rats with intermittent ethanol exposure after 72 h abstinence. Withdrawal signs emerge in rats with continuous ethanol exposure within 3 days after abstinence. Using behavioral tests, intraperitoneal injection of 7,8-DHF can reduce excessive ethanol consumption and preference as well as withdrawal signs in rats with repeated ethanol exposure. Interestingly, microinjection of K252a, an antagonist of TrkB, into the VTA blocks the effects of 7,8-DHF on ethanol-related behaviors. Furthermore, direct microinjection of BDNF into the VTA mimics the effect of 7,8-DHF on ethanol related behaviors. Taken together, 7,8-DHF attenuates alcohol-related behaviors in rats undergoing alcohol consumption via TrkB in the VTA. Our findings suggest BDNF-TrkB in VTA is a part of regulating signals for opposing neural adaptations in AUD, and 7,8-DHF may serve as a potential candidate for treating alcoholism.

Role of MyD88 in IL-1β and Ethanol Modulation of GABAergic Transmission in the Central Amygdala.

Myeloid differentiation primary response protein (MyD88) is a critical neuroimmune adaptor protein in TLR (Toll-like receptor) and IL-1R (Interleukin-1 receptor) signaling complexes. These two pro-inflammatory families play an important role in the neurobiology of alcohol use disorder, specifically MyD88 regulates ethanol drinking, ethanol-induced sedation, and ethanol-induced deficits in motor coordination. In this study, we examined the role of MyD88 in mediating the effects of IL-1β and ethanol on GABAergic transmission in the central amygdala (CeA) of male mice using whole-cell patch-clamp recordings in combination with pharmacological (AS-1, a mimetic that prevents MyD88 recruitment by IL-1R) and genetic (Myd88 knockout mice) approaches. We demonstrate through both approaches that IL-1β and ethanol’s modulatory effects at CeA GABA synapses are not dependent on MyD88. Myd88 knockout potentiated IL-1β’s actions in reducing postsynaptic GABAA receptor function. Pharmacological inhibition of MyD88 modulates IL-1β’s action at CeA GABA synapses similar to Myd88 knockout mice. Additionally, ethanol-induced CeA GABA release was greater in Myd88 knockout mice compared to wildtype controls. Thus, MyD88 is not essential to IL-1β or ethanol regulation of CeA GABA synapses but plays a role in modulating the magnitude of their effects, which may be a potential mechanism by which it regulates ethanol-related behaviors.

Novel Adenosine Analog, N6-(4-Hydroxybenzyl)-Adenosine, Dampens Alcohol Drinking and Seeking Behaviors.

Adenosine signaling is associated with ethanol-related behaviors. We previously found that adenosine A2A receptor (A2AR) activation dampens ethanol drinking behaviors in equilibrative nucleoside transporter 1 (ENT1) knockout mice, and A2AR inhibition augments reward-seeking behavior in wild-type mice. The novel adenosine analog N6-(4-hydroxybenzyl)-adenosine (NHBA), which is isolated from the rhizomes of Gastrodia elata, activates A2AR and inhibits ENT1. Here, we examined the effects of NHBA on ethanol drinking in the two-bottle choice test and operant ethanol seeking behaviors. We selected mice exhibiting high ethanol drinking behavior in the two-bottle choice test. NHBA (0.1 mg/kg, i.p.) reduced ethanol drinking behavior in a limited-access 3-hour drinking session in high-consumption ethanol drinking mice, and NHBA (0.1 mg/kg, i.p.) did not alter locomotor activity in the open-field test. Operant conditioning with 10% ethanol and 10% sucrose (10E10S) reward increased zone entries and time spent in the ethanol zone, while NHBA (0.1 mg/kg, i.p.) dampened ethanol zone preference in the Y-maze. Furthermore, NHBA (0.1 mg/kg, i.p.) devalued 10E10S and 10% ethanol (10E) reward after operant conditioning with 10E10S and 10E. Taken together, NHBA through A2AR activation and ENT1 modulation may dampen ethanol drinking and seeking behaviors, suggesting that NHBA is a potential therapeutic agent for treating alcohol use disorder. SIGNIFICANCE STATEMENT: Our work highlights that A2AR activation and ENT1 inhibition by a novel adenosine analog isolated from Gastrodia elata, N6-(4-hydroxybenzyl)-adenosine, decreases ethanol drinking and seeking behaviors. We suggest that NHBA is a potential therapeutic agent to treat alcohol use disorder.

Type 1 equilibrative nucleoside transporter (ENT1) regulates sex-specific ethanol drinking during disruption of circadian rhythms.

Disruptions in circadian rhythms are risk factors for excessive alcohol drinking. The ethanol-sensitive adenosine equilibrative nucleoside transporter type 1 (ENT1, slc29a1) regulates ethanol-related behaviors, sleep, and entrainment of circadian rhythms. However, the mechanism underlying the increased ethanol consumption in ENT1 knockout (KO) mice in constant light (LL) and whether there are sex differences in ethanol consumption in ENT1 mice are less studied. Here, we investigated the effects of loss of ENT1, LL, and sex on ethanol drinking using two-bottle choice. In addition, we monitored the locomotor activity rhythms. We found that LL increased ethanol drinking and reduced accumbal ENT1 expression and adenosine levels in male but not female mice, compared with control mice. Interestingly, only LL-exposed male, not female, ENT1 KO mice exhibited higher ethanol drinking and a longer circadian period with a higher amplitude compared with wild-type (WT) mice. Furthermore, viral-mediated rescue of ENT1 expression in the NAc of ENT1 KO mice reduced ethanol drinking, demonstrating a possible causal link between ENT1 expression and ethanol drinking in males. Together, our findings indicate that deficiency of ENT1 expression contributes to excessive ethanol drinking in a sex-dependent manner.

Voluntary ethanol consumption during early social isolation and responding for ethanol in adulthood.

Little is known about the influence of rearing environments concurrent with voluntary intermittent access to ethanol on subsequent adult ethanol-related behaviors. Previous research has shown that adult rats reared in post-weaning, social isolation conditions (IC) respond more for operant ethanol compared to laboratory standard conditions (SC). Ethanol-exposed adolescents tend to consume more ethanol in adulthood than rats exposed as adults. The current study examined voluntary ethanol consumption during adolescence between IC and SC rats, subsequent operant responding for ethanol, and extinction of responding in the same rats as adults. Differences in ethanol metabolism may alter the amount of reward value per unit of ethanol consumed. Therefore, the current study also examined blood ethanol concentrations (BEC) between IC rats and SC rats. Ethanol-naïve Long-Evans rats arrived in the lab at postnatal day (PND) 21 and were separated into either IC or SC where they remained for the duration of the experiments. On PND 27, rats received intermittent access to 20% ethanol (3 days/week) for 4 or 6 weeks. Rats in the 6-week cohort were then trained to lever press for 20% ethanol in 30-min sessions followed by extinction. A separate cohort was reared in IC or SC, injected with 1.5 or 3.0g/kg of ethanol (intraperitoneally [i.p.]), followed by BEC measurement. Overall, IC rats had higher ethanol preference and consumption during adolescence/early adulthood. IC and SC rats did not differ in their rates of operant responding for ethanol, and SC rats responded more than IC rats during extinction. There were no differences in BEC between IC and SC rats. These findings highlight the importance of the environment during rat adolescent development with isolation conditions increasing binge-like drinking and ethanol preference after 3-4 weeks without differences in metabolism as a potential factor. Additionally, the findings indicate that intermittent adolescent access to ethanol may change typical differences in operant responding patterns between IC and SC rats in adulthood.

Increasing Brain-Derived Neurotrophic Factor (BDNF) in medial prefrontal cortex selectively reduces excessive drinking in ethanol dependent mice

The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) has been implicated in a number of neuropsychiatric disorders, including alcohol use disorder. Studies have shown that BDNF activity in cortical regions, such as the medial prefrontal cortex (mPFC) mediates various ethanol-related behaviors. We previously reported a significant down-regulation in Bdnf mRNA in mPFC following chronic ethanol exposure compared to control mice. The present study was conducted to extend these findings by examining whether chronic ethanol treatment reduces BDNF protein expression in mPFC and whether reversing this deficit via direct injection of BDNF or viral-mediated overexpression of BDNF in mPFC alters voluntary ethanol consumption in dependent and nondependent mice. Repeated cycles of chronic intermittent ethanol (CIE) exposure was employed to model ethanol dependence, which produces robust escalation of ethanol intake. Results indicated that CIE treatment significantly increased ethanol intake and this was accompanied by a significant decrease in BDNF protein in mPFC that lasted at least 72 h after CIE exposure. In a separate study, once dependence-related increased drinking was established, bilateral infusion of BDNF (0, 0.25, 0.50 μg) into mPFC significantly decreased ethanol intake in a dose-related manner in dependent mice but did not affect moderate drinking in nondependent mice. In a third study, viral-mediated overexpression of BDNF in mPFC prevented escalation of drinking in dependent mice but did not alter intake in nondependent mice. Collectively, these results provide evidence that adaptations in cortical (mPFC) BDNF activity resulting from chronic ethanol exposure play a role in mediating excessive ethanol drinking associated with dependence.

Systemic blockade of LPA1/3 lysophosphatidic acid receptors by ki16425 modulates the effects of ethanol on the brain and behavior

The systemic administration of lysophosphatidic acid (LPA) LPA1/3 receptor antagonists is a promising clinical tool for cancer, sclerosis and fibrosis-related diseases. Since LPA1 receptor-null mice engage in increased ethanol consumption, we evaluated the effects of systemic administration of an LPA1/3 receptor antagonist (intraperitoneal ki16425, 20 mg/kg) on ethanol-related behaviors as well as on brain and plasma correlates. Acute administration of ki16425 reduced motivation for ethanol but not for saccharine in ethanol self-administering Wistar rats. Mouse experiments were conducted in two different strains. In Swiss mice, ki16425 treatment reduced both ethanol-induced sedation (loss of righting reflex, LORR) and ethanol reward (escalation in ethanol consumption and ethanol-induced conditioned place preference, CPP). Furthermore, in the CPP-trained Swiss mice, ki16425 prevented the effects of ethanol on basal c-Fos expression in the medial prefrontal cortex and on adult neurogenesis in the hippocampus. In the c57BL6/J mouse strain, however, no effects of ki16425 on LORR or voluntary drinking were observed. The c57BL6/J mouse strain was then evaluated for ethanol withdrawal symptoms, which were attenuated when ethanol was preceded by ki16425 administration. In these animals, ki16425 modulated the expression of glutamate-related genes in brain limbic regions after ethanol exposure; and peripheral LPA signaling was dysregulated by either ki16425 or ethanol. Overall, these results suggest that LPA1/3 receptor antagonists might be a potential new class of drugs that are suitable for treating or preventing alcohol use disorders. A pharmacokinetic study revealed that systemic ki16425 showed poor brain penetration, suggesting the involvement of peripheral events to explain its effects.

GluN2B Subunit of the NMDA Receptor: The Keystone of the Effects of Alcohol During Neurodevelopment.

The glutamatergic system plays a central role in both the acute and chronic effects of ethanol. Among all the glutamate receptors the ionotropic NMDA receptors are crucial because of their role in synaptic plasticity. A large body of evidences suggests that short-term and long-term effects of ethanol may change synaptic plasticity via an alteration of the expression of the GluN2B subunit, one constitutive element of the NMDA receptor. The present review is focusing on the role of the GluN2B subunit after ethanol exposure during early life (in utero and adolescence) and also at adulthood. The roles of other NMDA subunits are also discussed in the context of the increasing evidence that the ratio of the different subunits, such as GluN2A-to-GluN2B, seems to better reflect the effects of ethanol and to explain how ethanol exposure can have short lasting and long lasting effects on synaptic plasticity, cognitive processes and some of the ethanol-related behaviors.

The lateral hypothalamus to lateral habenula projection, but not the ventral pallidum to lateral habenula projection, regulates voluntary ethanol consumption

The lateral habenula (LHb) is an epithalamic brain region implicated in aversive processing via negative modulation of midbrain dopamine (DA) and serotonin (5-HT) systems. Given the role of the LHb in inhibiting DA and 5-HT systems, it is thought to be involved in various psychiatric pathologies, including drug addiction. In support, it has been shown that LHb plays a critical role in cocaine- and ethanol-related behaviors, most likely by mediating drug-induced aversive conditioning. In our previous work, we showed that LHb lesions increased voluntary ethanol consumption and operant ethanol self-administration and blocked yohimbine-induced reinstatement of ethanol self-administration. LHb lesions also attenuated ethanol-induced conditioned taste aversion suggesting that a mechanism for the increased intake of ethanol may be reduced aversion learning. However, whether afferents to the LHb are required for mediating effects of the LHb on these behaviors remained to be investigated. Our present results show that lesioning the fiber bundle carrying afferent inputs to the LHb, the stria medullaris (SM), increases voluntary ethanol consumption, suggesting that afferent structures projecting to the LHb are important for mediating ethanol-directed behaviors. We then chose two afferent structures as the focus of our investigation. We specifically studied the role of the inputs from the lateral hypothalamus (LH) and ventral pallidum (VP) to the LHb in ethanol-directed behaviors. Our results show that the LH-LHb projection is necessary for regulating voluntary ethanol consumption. These results are an important first step towards understanding the functional role of afferents to LHb with regard to ethanol consumption.

Environmental enrichment reduces chronic psychosocial stress-induced anxiety and ethanol-related behaviors in mice

Previous research from our laboratory has shown that exposure to chronic psychosocial stress increased voluntary ethanol consumption and preference as well as acquisition of ethanol-induced conditioned place preference (CPP) in mice. This study was done to determine whether an enriched environment could have “curative” effects on chronic psychosocial stress-induced ethanol intake and CPP. For this purpose, experimental mice “intruders” were exposed to the chronic subordinate colony (CSC) housing for 19 consecutive days in the presence of an aggressive “resident” mouse. At the end of that period, mice were tested for their anxiety-like behavior using the elevated plus maze (EPM) test then housed in a standard or enriched environment (SE or EE respectively). Anxiety and ethanol-related behaviors were investigated using the open field (OF) test, a standard two-bottle choice drinking paradigm, and the CPP procedure. As expected, CSC exposure increased anxiety-like behavior and reduced weight gain as compared to single housed colony (SHC) controls. In addition, CSC exposure increased voluntary ethanol intake and ethanol-CPP. Interestingly, we found that EE significantly and consistently reduced anxiety and ethanol consumption and preference. However, neither tastants' (saccharin and quinine) intake nor blood ethanol metabolism were affected by EE. Finally, and most importantly, EE reduced the acquisition of CPP induced by 1.5g/kg ethanol. Taken together, these results support the hypothesis that EE can reduce voluntary ethanol intake and ethanol-induced conditioned reward and seems to be one of the strategies to reduce the behavioral deficits and the risk of anxiety-induced alcohol abuse.

Viral-mediated overexpression of the Myelin Transcription Factor 1 (MyT1) in the dentate gyrus attenuates anxiety- and ethanol-related behaviors in rats.

Myelin Transcription Factor 1 (MyT1), a member of the Zinc Finger gene family, plays a fundamental role in the nervous system. Recent research has suggested that this transcription factor is associated with the pathophysiology of psychiatric disorders including addiction, schizophrenia, and depression. However, the role of MyT1 in anxiety- and ethanol-related behaviors is still unknown. We evaluated the effects of lentiviral-mediated overexpression of MyT1 in the dentate gyrus (DG) on anxiety- and ethanol-related behaviors in rats. We used the elevated plus maze (EPM) and the open field (OF) tests to assess anxiety-like behavior and a two-bottle choice procedure to measure the effects of MyT1 on ethanol intake and preference. MyT1 overexpression produced anxiolytic-like effects in the EPM test and decreased the number of fecal boli in the OF test, without affecting locomotor activity in both behavioral tests. Next, we demonstrated that ethanol intake and preference were decreased in the MyT1-overexpressing rats with no effect on saccharin and quinine, used to assess taste discrimination, and no effect on ethanol clearance suggesting specific alterations in the rewarding effects of ethanol. Most importantly, ectopic MyT1 overexpression increased both MyT1 and BDNF mRNA levels in the DG. Using Pearson's correlation, results showed a strong negative relationship between MyT1 mRNA and anxiety parameters and ethanol consumption and a positive correlation between MyT1 and BDNF mRNAs. Taken together, MyT1 along with being a key component in anxiety may be a suitable candidate in the search of the molecular underpinnings of alcoholism.