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Breast cancer remains a research priority due to its invasive phenotype. Although the role of ion channels in cancer is now well established, the role of inositol (1,4,5)-trisphosphate (IP3) receptors (IP3Rs) remains enigmatic. If the three IP3Rs subtypes expression have been identified in various cancers, little is known about their physiological role. Here, we investigated the involvement of IP3R type 3 (IP3R3) in the migration processes of three human breast cancer cell lines showing different migration velocities: the low-migrating MCF-7 and the highly migrating and invasive MDA-MB-231 and MDA-MB-435S cell lines. We show that a higher IP3R3 expression level, but not IP3R1 nor IP3R2, is correlated to a stronger cell line migration capacity and a sustained calcium signal. Interestingly, silencing of IP3R3 highlights an oscillating calcium signaling profile and leads to a significant decrease of cell migration capacities of the three breast cancer cell lines. Conversely, stable overexpression of IP3R3 in MCF-7 cells significantly increases their migration capacities. This effect is completely reversed by IP3R3 silencing. In conclusion, we demonstrate that IP3R3 expression level increases the migration capacity of human breast cancer cells by changing the calcium signature. ispartof: Oncotarget vol:8 issue:42 pages:72324-72341 ispartof: location:United States status: published

International audience; Cell morphology is altered in the migration process, and the underlying cytoskeleton remodeling is highly dependent of intracellular Ca2+ concentration. Many calcium channels are known to be involved in migration. Inositol 1,4,5-trisphosphate receptor (IP3R) was demonstrated to be implicated in breast cancer cells migration, but its involvement in morphological changes during the migration process remains unclear. In the present work, we showed that IP(3)R3 expression was correlated to cell morphology. IP(3)R3 silencing induced rounding shape and decreased adhesion in invasive breast cancer cell lines. Moreover, IP(3)R3 silencing decreased ARHGAP18 expression, RhoA activity, Cdc42 expression and (Y861)FAK phosphorylation. Interestingly, IP(3)R3 was able to regulate profilin remodeling, without inducing any myosin II reorganization. IP3R3 silencing revealed an oscillatory calcium signature, with a predominant oscillating profile occurring in early wound repair. To summarize, we demonstrated that IP3R3 is able to modulate intracellular Ca2+ availability and to coordinate the remodeling of profilin cytoskeleton organization through the ARHGAP18/RhoA/mDia1/PAK pathway.

Cell morphology is altered in the migration process, and the underlying cytoskeleton remodeling is highly dependent of intracellular Ca

Ethanol (EtOH)-induced behavioral sensitization (EIBS) is proposed to play a role in early and recurring steps of alcohol dependence, but its impact on alcohol abuse is not clear. EIBS development is dependent upon animal species, strain and also individual factors. We proposed here to decipher the co-expression of EIBS and EtOH intake in individual animals among outbred Swiss mice, which exhibit heterogeneity that parallels what may occur in humans. To do so, mice were exposed to a two-bottle choice with free access to water or 10% EtOH for 6 days just before and immediately after chronic intraperitoneal 2.5 g/kg ethanol injections once a day for 10 consecutive days. Based on their sensitization scores, mice were split into resistant and sensitized animals. First, we showed that individual susceptibility to EIBS is inversely correlated with voluntary EtOH consumption. Exposure to repeated EtOH during EIBS development increased subsequent EtOH intake among the entire population. Very interestingly, subsequent analyses suggested that the less the mice are sensitized the more they increase their EtOH intake; however, resistant mice were sensitive to EtOH adulteration with quinine, whereas sensitized ones maintained their EtOH intake levels, therefore exhibiting a compulsive-like drinking pattern. In addition, we showed that resistant mice do not exhibit a weaker sensitivity to the aversive properties of EtOH that may contribute to their higher level of EtOH intake compared to sensitized mice. This study confirms and extends previous data showing a deep relationship between propensity for EtOH consumption and susceptibility to EIBS in Swiss mice.

Adolescent alcohol binge drinking constitutes a major vulnerability factor to develop alcoholism. However, mechanisms underlying this susceptibility remain unknown. We evaluated the effect of adolescent binge-like ethanol intoxication on vulnerability to alcohol abuse in Sprague-Dawley rats. To model binge-like ethanol intoxication, every 2 days, rats received an ethanol injection (3.0 g/kg) for 2 consecutive days across 14 days either from postnatal day 30 (PND30) to 43 (early adolescence) or from PND 45 to PND 58 (late adolescence). In young adult animals, we measured free ethanol consumption in the two-bottle choice paradigm, motivation for ethanol in the operant self-administration task and both ethanol's rewarding and aversive properties in the conditioned place preference (CPP) and taste aversion (CTA) paradigms. While intermittent ethanol intoxications (IEI) during late adolescence had no effect on free-choice 10% ethanol consumption, we found that IEI during early adolescence promoted free-choice 10% ethanol consumption, enhanced motivation for ethanol in the self-administration paradigm and induced a loss of both ethanol-induced CPP and CTA in young adults. No modification in either sucrose self-administration or amphetamine-induced CPP was observed. As the nucleus accumbens (Nac) is particularly involved in addictive behavior, we analyzed IEI-induced long-term neuroadaptations in the Nac using c-Fos immunohistochemistry and an array of neurotransmission-related genes. This vulnerability to ethanol abuse was associated with a lower c-Fos immunoreactivity in the Nac and enduring alterations of the expression of Penk and Slc6a4, 2 neurotransmission-related genes that have been shown to play critical roles in the behavioral effects of ethanol and alcoholism.

Background Behavioral sensitization induced by repeated ethanol (EtOH) exposure may play a critical role in the development of alcohol dependence. Because recent data demonstrate that histone deacetylase inhibitor (HDACi) may be of interest in the treatment of addiction, we explored the effect of the HDACi sodium butyrate (NaB) on EtOH-induced behavioral sensitization (EIBS) in DBA/2J mice. We also investigated gene regulations in the striatum of sensitized mice using epigenetic- and signal transduction-related PCR arrays. Methods Mice were injected with saline or EtOH (0.5 to 2.5 g/kg) once a day for 10 days. Mice received NaB (200 to 600 mg/kg) 30 minutes before each injection (prevention protocol) or once daily between days 11 and 16 (reversal protocol). At day 17, brains were removed 30 minutes after a saline or EtOH challenge to assess gene and proteins levels. Results Only the intermediate EtOH doses (1.0 and 2.0 g/kg) were effective in inducing EIBS, and both doses were associated with specific gene regulations in the striatum. The induction of sensitization by 1.0 g/kg (but not 2.0 g/kg) EtOH was dose-dependently prevented or reversed by NaB. Among the 168 studied genes, EIBS blockade was associated with specific gene regulations (bcl-2, bdnf, hdac4, pak1, penk, tacr1, vip…) and changes in brain-derived neurotrophic factor in both striatum and prefrontal cortex. Conclusions These results indicate that EIBS is associated with specific gene regulations in the striatum depending on the EtOH dose and that NaB can be useful in blocking some long-lasting neuro-adaptations to repeated EtOH administrations.

Brain-derived neurotrophic factor (BDNF) within the striatum is part of a homeostatic pathway regulating alcohol consumption. Memantine, a non-competitive antagonist of N-methyl-D-aspartate receptors, induces expression of BDNF in several brain regions including the striatum. We hypothesized that memantine could decrease ethanol (EtOH) consumption via activation of the BNDF signalling pathway. Effects of memantine were evaluated in Long-Evans rats self-administering moderate or high amounts of EtOH 6, 30 and 54 hours after an acute injection (12.5 and 25 mg/kg). Motivation to consume alcohol was investigated through a progressive ratio paradigm. The possible role for BDNF in the memantine effect was tested by blockade of the TrkB receptor using the pharmacological agent K252a and by the BDNF scavenger TrkB-Fc. Candidate genes expression was also assessed by polymerase chain reaction array 4 and 28 hours after memantine injection. We found that memantine decreased EtOH self-administration and motivation to consume EtOH 6 and 30 hours post-injection. In addition, we found that inhibition or blockade of the BDNF signalling pathway prevented the early, but not the delayed decrease in EtOH consumption induced by memantine. Finally, Bdnf expression was differentially regulated between the early and delayed timepoints. These results demonstrate that an acute injection of memantine specifically reduces EtOH self-administration and motivation to consume EtOH for at least 30 hours. Moreover, we showed that BDNF was responsible for the early effect, but that the delayed effect was BDNF-independent.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.

International audience; The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is known to counteract in vitro the deleterious effects of toxic agents on cerebellar granule cell survival and differentiation. The potent antiapoptotic action of PACAP is mediated through inhibition of caspase-3 activity; however, additional proteins are likely involved and remain to be identified. Two-dimensional gel electrophoresis analysis coupled with mass spectrometry characterization led to the identification of a protein, peroxiredoxin 2, which was induced after a 6-h treatment with PACAP. Western blot analysis confirmed the regulation of peroxiredoxin 2 by PACAP and revealed that this protein is induced by both cyclic AMP and protein kinase C stimulators. Inhibition of peroxiredoxin 2 expression, using two distinct small-interfering RNAs (siRNAs), reduced the effect of PACAP on caspase-3 activity and cerebellar granule cell survival. Peroxiredoxin 2 expression was also induced in vivo and in vitro by ethanol. Although ethanol and PACAP exert opposite effects on caspase-3 activity, inhibition of peroxiredoxin 2 expression, using siRNAs, only reduced the ability of PACAP to prevent ethanol-induced caspase-3 activity. Taken together, these data indicate that peroxiredoxin 2 is probably involved in the neurotrophic effect of PACAP and suggest that this protein may have a therapeutic potential for the treatment of some neurodegenerative diseases

Ethanol (EtOH)-induced behavioral sensitization (EIBS) is defined as an enhancement of locomotor activity following repeated EtOH exposure and is proposed to reflect an increase in EtOH “wanting”. However, the reliability of the sensitization model in studying addiction is still a matter of debate. One major criticism is that the increase in locomotion occurring during sensitization may be a by-product of tolerance to the ataxic and/or sedative effects of EtOH. We investigated the relationship between EIBS amplitude and sensitivity to EtOH-induced ataxia and sedation after the development of tolerance to EtOH depressant effects in adult female DBA/2J mice. After receiving daily injection of saline or 2 g/kg EtOH during 10 consecutive days to induce EIBS, recovery from acute motor incoordination produced by ethanol (2 g/kg EtOH using rotarod) and from loss of righting reflex (4 g/kg EtOH) was measured. We showed that induction of EtOH sensitization after repeated administration of EtOH is associated with a more rapid recovery from acute motor incoordination and from sedation produced by ethanol when compared to the acute groups, suggesting the development of tolerance to the ataxic and sedative effects of EtOH. However, correlational analyses failed to detect any relationship between EIBS amplitude and the response to EtOH ataxic or sedative effects. Altogether, our results confirm and extend previous data showing a tolerance to the ataxic and sedative properties of EtOH after repeated exposure to EtOH and suggest that this tolerance is not related to the amplitude of EIBS.

International audience; Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cere-bellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H 2 O 2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cere-bral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.

Ethanol (EtOH)-induced behavioral sensitization (EIBS) is proposed to play a role in early and recurring steps of alcohol dependence, but its impact on alcohol abuse is not clear. EIBS development is dependent upon animal species, strain and also individual factors. We proposed here to decipher the co-expression of EIBS and EtOH intake in individual animals among outbred Swiss mice, which exhibit heterogeneity that parallels what may occur in humans. To do so, mice were exposed to a two-bottle choice with free access to water or 10% EtOH for 6 days just before and immediately after chronic intraperitoneal 2.5 g/kg ethanol injections once a day for 10 consecutive days. Based on their sensitization scores, mice were split into resistant and sensitized animals. First, we showed that individual susceptibility to EIBS is inversely correlated with voluntary EtOH consumption. Exposure to repeated EtOH during EIBS development increased subsequent EtOH intake among the entire population. Very interestingly, subsequent analyses suggested that the less the mice are sensitized the more they increase their EtOH intake; however, resistant mice were sensitive to EtOH adulteration with quinine, whereas sensitized ones maintained their EtOH intake levels, therefore exhibiting a compulsive-like drinking pattern. In addition, we showed that resistant mice do not exhibit a weaker sensitivity to the aversive properties of EtOH that may contribute to their higher level of EtOH intake compared to sensitized mice. This study confirms and extends previous data showing a deep relationship between propensity for EtOH consumption and susceptibility to EIBS in Swiss mice.

Brain-derived neurotrophic factor (BDNF) within the striatum is part of a homeostatic pathway regulating alcohol consumption. Memantine, a non-competitive antagonist of N-methyl-D-aspartate receptors, induces expression of BDNF in several brain regions including the striatum. We hypothesized that memantine could decrease ethanol (EtOH) consumption via activation of the BNDF signalling pathway. Effects of memantine were evaluated in Long-Evans rats self-administering moderate or high amounts of EtOH 6, 30 and 54 hours after an acute injection (12.5 and 25 mg/kg). Motivation to consume alcohol was investigated through a progressive ratio paradigm. The possible role for BDNF in the memantine effect was tested by blockade of the TrkB receptor using the pharmacological agent K252a and by the BDNF scavenger TrkB-Fc. Candidate genes expression was also assessed by polymerase chain reaction array 4 and 28 hours after memantine injection. We found that memantine decreased EtOH self-administration and motivation to consume EtOH 6 and 30 hours post-injection. In addition, we found that inhibition or blockade of the BDNF signalling pathway prevented the early, but not the delayed decrease in EtOH consumption induced by memantine. Finally, Bdnf expression was differentially regulated between the early and delayed timepoints. These results demonstrate that an acute injection of memantine specifically reduces EtOH self-administration and motivation to consume EtOH for at least 30 hours. Moreover, we showed that BDNF was responsible for the early effect, but that the delayed effect was BDNF-independent.

International audience; BACKGROUND: Ethanol-induced behavioral sensitization (EIBS) is proposed to play a role in early and recurring steps of addiction. EIBS does not occur uniformly in all animals even from the same inbred strain. Since recent data demonstrate that epigenetic mechanisms are likely to be involved in the development and the persistence of ethanol-related behaviors, we explored the involvement of epigenetic mechanisms in ethanol response after EIBS development. METHODOLOGY: DBA/2J mice were i.p. injected with saline or ethanol (2 g/kg) once a day for 10 consecutive days. At day 17, ethanol-treated mice were split in resistant and sensitized groups. Brains were then removed 30 min after a saline or 2 g/kg ethanol challenge to assess i) gene expression using PCR array targeting 84 epigenetic-related genes and ii) histone deacetylases (HDAC), histone acetylases (HAT) and DNA methyltransferases (DNMT) activities as well as H4K12 acetylation. PRINCIPAL FINDINGS: Acute ethanol administration decreased dnmt1, esco2 and rps6ka5 genes expression. These genes were similarly altered in sensitized but not in resistant mice after an ethanol challenge, suggesting that resistant mice were tolerant to the transcriptional outcomes of an ethanol challenge. Whereas global HAT or DNMT activity was not affected, global HDAC activity was reduced after an acute ethanol injection. HDAC inhibition occurred in all ethanol-treated mice but with a lesser extent in sensitized animals. As a consequence, H4 acetylation was specifically potentiated in the core of the Nac proportionally to the striatal HDAC activity decrease. CONCLUSIONS/SIGNIFICANCE: The present study highlights that the contrasted behavioral response to an ethanol challenge between resistant and sensitized mice may be mediated by epigenetic mechanisms occurring specifically in the striatum. Here we show that vulnerability to ethanol dependence and relapse could be, at least in part, due to individual variability in acute ethanol-induced epigenetic response.

International audience; The developing rat cerebellum is particularly sensitive to alcohol at the end of the first postnatal week, a period of intense neurogenesis. The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) has previously been shown to prevent the death of cultured neurons in vitro. We have thus investigated the capacity of PACAP to counteract ethanol toxicity in 8-day-old rats. Behavioral studies revealed that PACAP reduces the deleterious action of alcohol in the negative geotaxis test. Administration of ethanol induced a transient increase of the expression of pro-apoptotic genes including c-jun or caspase-3 , which could be partially blocked by PACAP. Alcohol inhibited the expression of the α6 GABA ( A ) subunit while PACAP increased neuroD2 mRNA level, two markers of neuronal differentiation. Although gene regulations occurred rapidly, a third injection of ethanol was required to strongly reduce the number of granule cells in the internal granule cell layer, an effect which was totally blocked by PACAP. The action of PACAP was mimicked by D-JNKi1 and Z-VAD-FMK, indicating the involvement of the jun and caspase-3 pathways in alcohol toxicity. The present data demonstrate that PACAP can counteract in vivo the deleterious effect of ethanol. The beneficial action of PACAP on locomotor activity precedes its activity on cell survival, indicating that PACAP can block the detrimental action of ethanol on cell differentiation.

International audience; Pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide of the vasoactive intestinal polypep-tide (VIP)-glucagon superfamily, was initially characterized by virtue of its ability to stimulate cAMP formation in cultured rat anterior pituitary cells. Three PACAP receptors have been cloned so far: a PACAP-selective receptor, termed PAC1-R, and two PACAP/VIP common receptors, termed VPAC1-R and VPAC2-R. PACAP and its receptors are widely expressed in the brain and in peripheral organs, notably in the hypothalamus and in endocrine glands. Indeed, there is now clear evidence that PACAP exerts neuroendocrine, paracrine, and autocrine control of the activity of the pituitary, thyroid, testis, ovary, adrenal medulla, adrenal cortex, and endocrine pancreas. These observations suggest that selective PACAP agonists and antagonists could have therapeutic value for the treatment of various endo-crine disorders.

S31.1 ADOLESCENT INTERMITTENT ETHANOL-INDUCED HISTONE MODIFICATIONS AND DNA DEMETHYLATION: ROLE IN ANXIETY AND ALCOHOLISM {#article-title-2} Histone modifications and DNA methylation/demethylation regulate gene expression and play an important role in synaptic plasticity. We investigated the effects of adolescent intermittent ethanol (AIE) treatment on epigenetic modifications in the amygdala and on anxiety-like behaviors in adolescent and adult rats. The effects of AIE [2 g/kg, intraperitoneal; 2-days on/2-days off, 4 cycles (8 injections), postnatal days 28-41] on anxiety-like behaviors in adolescent rats was measured during withdrawal and effects of AIE exposure on anxiety-like and alcohol drinking behaviors at adulthood (postnatal day 92) were measured. Adolescent rats displayed anxiety-like behaviors after 24 hrs of ethanol withdrawal. At this time point, amygdaloid nuclear and cytosolic HDAC activity and levels of HDAC2 and HDAC4 were increased and histone (H3-K9) acetylation in the central (CeA) and medial amygdala (MeA) was decreased. Interestingly, in AIE adult rats nuclear HDAC activity and HDAC2 protein levels remained increased and histone acetylation remained decreased. We also found decreased histone H3 acetylation in promoters of Arc, NPY, and various BDNF exons in the amygdala of AIE compared with control (adolescent intermittent saline) adult rats. AIE produced a reduction in dendritic spines in the CeA and MeA and decreased mRNA levels of GADD45g, but not GADD45a or GADD45b in the amygdala of adult rats. AIE induced anxiety-like and alcohol drinking behaviors at adulthood were attenuated by treatment with trichostatin A. These results suggest that AIE induced epigenetic changes in the amygdala may play a crucial role in anxiety-like and alcohol-drinking behaviors at adulthood (supported by NADIA grant from NIH-NIAAA). # S31.2 IS THERE A FUTURE FOR USING HDAC INHIBITORS IN ALCOHOL DEPENDENCE? {#article-title-3} Emerging evidence suggests that epigenetic alterations to the genome, including DNA methylation and histone modifications, are important mechanisms underlying alcohol addiction. With the new field of epigenetics, there is now the opportunity to integrate the role of the epigenome not only in the short-term effects of alcohol but also in the enduring neuro-adaptations caused by chronic use of alcohol. The epigenetic modifications may be part of the neuro-adpatations occurring during the transition from the controlled intake to the loss of control and may explain how alcohol can have such a persistent effect on brain gene expression and functioning. There are now accumulating data showing that pharmacological tools targeting enzymes involved in epigenetic modifications may be useful in reducing or preventing some behavioral responses to alcohol. In the present work we have investigated the effects of different HDAC inhibitors (sodium butyrate, trichostatine A, MS-275 and sirtinol) on alcohol intake in rats with several paradigms, i.e. operant 10% ethanol self-administration, intermittent 20% ethanol intake and alcohol deprivation effect. We have also investigated the effects of HDAC inhibitors on excessive alcohol intake in alcohol-dependent animals and explored associated modifications in the brain levels of acetylated histone H3. Finally we tested the effects of HDAC inhibitors on ethanol-induced behavioral locomotor sensitization in mice and analyzed alterations in striatal gene expression and both HDAC and HAT activities. Altogether our results show that HDAC inhibitor can reduce and / or prevent several behavioral effects of alcohol and that this effect of HDAC inhibitors can be due, at least in part, to epigenetic mechanism. # S31.3 GENETIC AND ENVIRONMENTAL RISKS FOR PATTERNS OF ALCOHOL USE FROM ADOLESCENCE THROUGH EARLY ADULTHOOD {#article-title-4} Purpose. Early and heavy initiation of alcohol use is a risk for later abuse and health problems; hence, identifying contributors to youth alcohol use is a central goal. This paper seeks to delineate trajectories of alcohol use and abuse from early adolescence through early adulthood, and identify environmental and genetic risk factors contributing to problematic patterns of early alcohol use. We compare four leading explanations for problematic alcohol use: social norms, social controls, stress, and genetic predispositions. Methods. Data were drawn from a nationally representative study of American youth (Add Health), following 20,745 youth from early adolescence through early adulthood. Youth reported on alcohol use and inebriation. Youth, parents, peers, and school administrators reported on social norms (parent drinking, peer drinking, schoolmate drinking), social controls (parental supervision, monitoring, school punishment), and stressful life events. A genetic risk score was calculated using DNA data on three dopaminergic polymorphisms (DRD4, DAT1, MAOA). Results. Trajectory analysis found five patterns of alcohol use from early adolescence through early adulthood, delineating abstainers, low users, early declining, early increasing, and late increasing patterns of alcohol use. Analyses identified four trajectories of inebriation: abstainers, low users, early declining, and increasing. Multilevel multinomial logistic regression models predicted patterns of drinking and inebriation, finding significant effects of social norms, social control, and life stressors for males' patterns of alcohol use and inebriation. For females, social norms, life stressors, and genetic risks were significant predictors of alcohol use and inebriation. Models found little evidence of gene by environment interactions. # S31.4 PRENATAL ETHANOL-INDUCED HYPERMETHYLATION OF PROOPIOMELANOCORTIN GENE TRANSMITS THROUGH GERMLINE {#article-title-5} We have recently shown that alcohol exposure during the fetal period permanently suppresses the expression of stress controlling beta-endorphin due to hypermethylation of it's precursor POMC gene. In this study, we determined the mechanism by which the POMC gene is hypermethylated. Additionally, transgenerational studies were conducted to evaluate the germline-transmitted effect of alcohol. We found that fetal alcohol exposure decreased the levels of the activation histone marks H3K4, aceH3K9 and pH3S10 in POMC neurons, and increased the repressive histone mark H3K9. The fetal alcohol exposure also altered the protein levels and gene expression of POMC, Set7/9, CBP, HDAC2, G9a, Setdb1, MeCP2 and Dnmt1 except for Dnmt3a. Alcohol treatment also reduced the activation mark H3K4me3 along Exon 3 of POMC gene. Suppression of histone deacetylation and DNA methylation normalized POMC expression and functional abnormalities. Gestational choline supplementation also reversed alcohol-induced alteration in histone-modifying and DNA-methylating enzymes and normalized POMC gene promoter methylation, POMC gene expression and β-endorphin peptide production in POMC neurons. Fetal alcohol-induced POMC gene methylation, expression and functional defects persisted in the F2 and F3 male but not in female germline. Additionally, the hypermethylated POMC gene was detected in sperms of fetal alcohol exposed F1 offspring that was transmitted through F3 generation via male germline. Overall, these research findings collectively demonstrate that epigenetic changes contribute to the effects of fetal alcohol exposure on POMC gene expression as well as the perpetuation of abnormal POMC neuronal functions through subsequent generations via the male. (Supported by NIH Grant AA016695 and R37 AA08757).