Juvenile variable stress modulates, in female but not in male Wistar rats, ethanol intake in adulthood.

Early stress can increase vulnerability to psychopathological disorders, including substance use disorders. The effects of stress in the juvenile period of the rat, that extends between weaning and the onset of adolescence (equivalent to late human childhood), have received little attention. This study assessed short and long-term behavioral effects of juvenile stress, with a focus on effects on ethanol intake. Male and female Wistar rats were exposed to variable stress (restraint, elevated platform, forced swimming, and social instability) or to restraint stress only, between postnatal days 26 to 29 (PDs 26-29). During adolescence, patterns of anxiety (PD 31) and depression (PD 33), ethanol intake (PDs 36-45) and behavioral sensitivity to the effects of acute stress (PD 47) were evaluated. In adulthood, alcohol ingestion was assessed through two-bottle ethanol intake tests (PDs 75-85). An additional experiment measured blood ethanol levels after a limited access intake session in adolescence. Exposure to juvenile variable stress exerted very mild effects in adolescence, but reduced ethanol ingestion in adulthood, in females only. Ethanol intake during the limited access session was significantly correlated to blood alcohol levels. The results indicate that a schedule of juvenile variable stress that did not significantly alter anxiety-related behaviors induced, nonetheless, sexually dimorphic effects on ethanol intake in adulthood. Early stress exposure that reduced alcohol intake in Wistar rats has been associated with changes on brain opioid and dopamine receptors. These results highlight the impact of early stress exposure on adult female ethanol consumption and its possible underlying neurobiological changes, involving opioid and dopamine receptors.

Folate administration ameliorates neurobehavioral effects of prenatal ethanol exposure.

Background: Prenatal ethanol exposure (PEE) induces heightened ethanol intake at adolescence in preclinical studies. Ethanol intake alters the absorption of folate, a methyl-group donor critical for numerous cellular functions. The prenatal administration of folate is, therefore, a promising approach to reduce the effects of PEE.Objectives: Experiment 1 determined if prenatal folate modulated the effects of PEE on ethanol intake, anxiety-like response, and exploratory behaviors (Experiment 1) in Wistar rats. Experiment 2 assessed, in rats not given PEE, if postnatal folate reversed effects of ethanol exposure at postnatal days 28-42. Experiment 3 assessed if folate altered blood ethanol levels (BELs).Methods: Experiment 1 involved 242 (125 male) adolescent Wistar rats derived from dams given folate (20 mg/kg, gestational days - GD- 13-20) + ethanol (2.0 g/kg, GD 17-20), ethanol, or vehicle only at pregnancy. Experiment 2 involved 29 male adolescents administered vehicle or ethanol doses co-administered or not with folate. In Experiment 3 twelve adult females were tested for BELs after folate administration. These tests were applied: intake tests, light dark box (LDB), elevated plus maze, open field and concentric square field.Results: PEE heightened ethanol intake (η2 ps = 0.06-07) and induced hyperactivity and a reduced latency to exit the white area of the LDB (η2 ps = 0.12-17). These effects were partially inhibited by folate (p > .05). Rats exposed to ethanol exposure at adolescence exhibited reduced motor activity (η2 p = .17), regardless of folate treatment. Folate did not affect BELs.Conclusion: Folate administration should be considered as a preventive or acute treatment to attenuate the neurobehavioral effects of PEE.

Binge eating promotes ethanol self-administration in female rats with a history of intermittent ethanol exposure at adolescence.

BACKGROUND: Ethanol drinking begins during adolescence and, particularly when occurs in a binge-like pattern, exerts lingering adverse consequences. Pre-clinical studies indicate that intermittent ethanol exposure (IEA, a model of repeated ethanol intoxication), or binge eating (BE) can increase subsequent ethanol consumption. It is unknown if the promoting effects of BE upon ethanol drinking are found in female rats and are modulated by IEA at adolescence. This study assessed interactive effects between IEA and BE, upon ethanol drinking. METHODS: Female Wistar rats were given 4.0 g/kg ethanol, every other day from postnatal day 25-45. At adulthood, they were exposed to sessions in which a brief offering of a sizeable portion of highly palatable sugary pills was followed by a 120-min exposure to an ethanol bottle. RESULTS: Exploratory activity and recognition memory was not affected by the IEA. Glutathione peroxidase and catalase activity, and lipid peroxidation (measured in blood and brain at the end of the procedure) were not significantly affected by IEA or BE exposure. BE alone had a mild promoting effect on ethanol ingestion. Those rats that underwent IEA and BE, however, exhibited heightened and sustained ethanol self-administration (average of 2.12 g/kg/120 min, vs 1.15 g/kg/120 min of the other groups), that persisted throughout the BE sessions. IEA and a history of BE also promoted ethanol intake or preference in a two-bottle endpoint test. CONCLUSION: The study suggests that exposure to IEA exerts, when followed by BE at adulthood, promoting effects upon ethanol intake, particularly at concentrations ≥ 6%.

Transient serotonin depletion at adolescence, but not at early infancy, reduced subsequent anxiety-like behavior and alcohol intake in female mice.

RATIONALE: Serotonin (5-HT) plays an important role in the organization of the central nervous system and in the development of social interaction deficits and psychiatric disorders, including anxiety, depression, and addiction disorders. Notably, disruption of the 5-HT system during sensitive periods of development exerts long-term consequences, including altered anxiety response and problematic use of alcohol. OBJECTIVE: we analyzed, in mice, the effects of transient 5-HT depletion at infancy or adolescence on subsequent anxiety-like behavior and alcohol intake during adolescence. METHODS: C57/BL6 male and female mice were administered a 5-HT synthesis inhibitor (PCPA; 4-chloro-DL-phenylalanine methyl ester hydrochloride) at infancy (postnatal days 14-16 [PD14-16]) or adolescence (PD40-42). Eleven (± 1) days after treatment, mice were assessed for ethanol intake in daily two-bottle choice tests and for anxiety response via the elevated plus maze. RESULTS: Female, but not male, mice transiently depleted of 5-HT at adolescence (but not those depleted at the perinatal stage) exhibited a significant reduction in anxiety response, which was accompanied by a significant reduction on alcohol intake. CONCLUSION: Transient 5-HT inhibition at adolescence may act, in females, as a protective factor for the emergence of anxiety disorders and problematic use of alcohol during adolescence.

Environmental enrichment during adolescence heightens ethanol intake in female, but not male, adolescent rats that are selectively bred for high and low ethanol intake during adolescence.

Background: Discriminating between adolescents who will eventually have ethanol use problems from those who do not is important. Environmental enrichment is a promising approach to reduce drug-related problems, but its impact on ethanol's effects and intake is being scrutinized. Objective: We tested the effects of environmental enrichment on ethanol intake, preference, and anxiety-like response as well as shelter seeking and risk-taking behaviors. Methods: Experiment 1 examined ethanol intake, preference, and anxiety-like responses in 46 male and 54 female Wistar rats that were derived from a short-term breeding program that selected for high and low ethanol drinking during adolescence (ADHI2 and ADLO2 lines, respectively). Shelter-seeking and risk-taking behaviors were assessed (Experiment 2) in ADHI2 and ADLO2 rats (73 males, 76 females) reared under environmental enrichment or standard housing conditions and given doses of ethanol (2.5 g/kg, intraperitoneal) for 3 weeks. Environmental enrichment was applied on postnatal days 21-42. Ethanol intake was measured on postnatal days 42-68. Anxiety-like behavior and exploratory responses were assessed using the light-dark box and multivariate concentric square field test. Results: In Experiment 1, environmental enrichment increased ethanol intake in female, but not male, ADHI2 and ADLO2 rats (p < 0.05). In the baseline measurement of Experiment 2, ADHI2 rats exhibited reduced risk-taking and increased anxiety-like behavior (p < .05). After exposure to environmental enrichment the ADHI and ADLO rats, both males and females, exhibited increased risk-taking and exploratory behavior (p < 0.05). Conclusions: Environmental enrichment appears to increase ethanol intake in female rats by promoting the exploration of new environments or stimuli. The findings indicate that environmental enrichment increased ethanol intake in female, but not male, rats. Clinical programs that treat alcohol use disorder by emphasizing environmental stimulation should be designed with caution.

Selective alterations in endogenous opioid system genes expression in rats selected for high ethanol intake during adolescence.

Historically, the roots of alcoholism have been linked to either environment or heredity. However, the interaction between these factors is still largely unexplored. The evidence supports a link between alcohol consumption and the endogenous opioid system. We here studied the opioid genes expression in male and female Wistar rats derived from a short-term breeding program which selected -- at adolescence -- for high (ADHI line) or low (ADLO line) ethanol drinking. Specifically, in this work we analyzed central opioid gene expression in the rats of the second filial generation (S2-ADLO and S2-ADHI). Selective downregulation of pronociceptin (Pnoc) and its receptor (Oprl1) mRNA levels were observed in the prefrontal cortex of male S2-ADHI rats when compared to S2-ADLO, and for Oprl1 also in the nucleus accumbens. An increase in gene expression was instead observed for pro-opiomelanocortin (Pomc) in the nucleus accumbens of S2-ADHI males when compared to S2-ADLO, as well as for mu opioid receptor (Oprm1) but in females. The differences in mRNA levels may be due to the different alcohol consumption between the two groups of rats or may represent pre-existing differences between them. Moreover, we show a sex-specific modulation of the expression of these genes, thus pointing out the importance of sex on ethanol responses. The results might lead to more specific and effective pharmacological treatments for alcoholism.

MeCP2 Deficiency Disrupts Kainate-Induced Presynaptic Plasticity in the Mossy Fiber Projections in the Hippocampus.

Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events in vivo in the hippocampus of MeCP2 mutant mice. While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.

Anxiety response and restraint-induced stress differentially affect ethanol intake in female adolescent rats.

Anxiety disorders are more likely to occur in women than in men, usually emerge during adolescence and exhibit high comorbidity with alcohol use disorders (AUD). Adolescents with high levels of anxiety or heightened reactivity to stress may be at-risk for developing AUD. An approach to analyze if high levels of inborn anxiety predict greater ethanol drinking is to assess the latter variable in subjects classified as high- or low-anxiety responders. The present study assessed ethanol drinking in adolescent, female Wistar, rats classified as high-, low- or average-anxiety responders and exposed or not to restraint stress (RS, Exp. 1). Classification was made through a multivariate index derived from testing anxiety responses in an elevated plus maze and a light-dark box tests. RS was applied after animals had been initiated to ethanol drinking. Intake of sweetened ethanol was unaffected by level of anxiety response. Adolescents with high levels of inborn anxiety exhibited significantly higher intake of unsweetened ethanol than counterparts with standard levels of anxiety, yet this effect was inhibited by RS exposure. Experiment 2 assessed FOS immunoreactivity after RS. Stress induced a significant increase in FOS immunoreactivity at the paraventricular nucleus, yet this effect was unaffected by level of anxiety response. Female adolescents with high levels of basal anxiety may be at-risk for exhibiting increased predisposition for ethanol intake and preference. The study also indicates that stress may exert differential effects on adolescent ethanol intake as a function of the level of anxiety response.

Age-related effects of chronic restraint stress on ethanol drinking, ethanol-induced sedation, and on basal and stress-induced anxiety response.

Adolescents are sensitive to the anxiolytic effect of ethanol, and evidence suggests that they may be more sensitive to stress than adults. Relatively little is known, however, about age-related differences in stress modulation of ethanol drinking or stress modulation of ethanol-induced sedation and hypnosis. We observed that chronic restraint stress transiently exacerbated free-choice ethanol drinking in adolescent, but not in adult, rats. Restraint stress altered exploration patterns of a light-dark box apparatus in adolescents and adults. Stressed animals spent significantly more time in the white area of the maze and made significantly more transfers between compartments than their non-stressed peers. Behavioral response to acute stress, on the other hand, was modulated by prior restraint stress only in adults. Adolescents, unlike adults, exhibited ethanol-induced motor stimulation in an open field. Stress increased the duration of loss of the righting reflex after a high ethanol dose, yet this effect was similar at both ages. Ethanol-induced sleep time was much higher in adult than in adolescent rats, yet stress diminished ethanol-induced sleep time only in adults. The study indicates age-related differences that may increase the risk for initiation and escalation in alcohol drinking.

Prenatal ethanol increases ethanol intake throughout adolescence, alters ethanol-mediated aversive learning, and affects µ but not δ or κ opioid receptor mRNA expression.

Animal models of prenatal ethanol exposure (PEE) have indicated a facilitatory effect of PEE on adolescent ethanol intake, but few studies have assessed the effects of moderate PEE throughout adolescence. The mechanisms underlying this facilitatory effect remain largely unknown. In the present study, we analysed ethanol intake in male and female Wistar rats with or without PEE (2.0 g/kg, gestational days 17-20) from postnatal days 37 to 62. The results revealed greater ethanol consumption in PEE rats than in controls, which persisted throughout adolescence. By the end of testing, ethanol ingestion in PEE rats was nearly 6.0 g/kg. PEE was associated with insensitivity to ethanol-induced aversion. PEE and control rats were further analysed for levels of µ, δ and κ opioid receptor mRNA in the infralimbic cortex, nucleus accumbens shell, and ventral tegmental area. Similar levels of mRNA were observed across most areas and opioid receptors, but µ receptor mRNA in the ventral tegmental area was significantly increased by PEE. Unlike previous studies that assessed the effects of PEE on ethanol intake close to birth, or in only a few sessions during adolescence, the present study observed a facilitatory effect of PEE that lasted throughout adolescence. PEE was associated with insensitivity to the aversive effect of ethanol, and increased levels of µ opioid receptor transcripts. PEE is a prominent vulnerability factor that probably favors the engagement of adolescents in risky trajectories of ethanol use.

Binge ethanol intoxication heightens subsequent ethanol intake in adolescent, but not adult, rats.

A question still to be answered is whether ethanol initiation has a greater effect on ethanol consumption if it occurs during adolescence than in adulthood. This study assessed the effect of ethanol initiation during adolescence or adulthood on voluntary ethanol consumption when animals were still within the same age range. Adolescent or adult rats were given 5, 2, or 0 ethanol exposures. The animals were tested for ethanol consumption through two-bottle choice tests, before undergoing a 1-week deprivation. A two-bottle assessment was conducted after the deprivation. Adolescents, but not adults, given two ethanol administrations during initiation exhibited significantly higher ethanol intake during the pre-deprivation period. These adolescents also exhibited a threefold increase in ethanol intake after 7 days of drug withdrawal, when compared with controls. These findings suggest that very brief experience with binge ethanol intoxication in adolescence, but not in adulthood, impacts later predisposition to drink.

Maternal isolation during the first two postnatal weeks affects novelty-induced responses and sensitivity to ethanol-induced locomotor activity during infancy.

Animals exposed to chronic maternal separation (MS) exhibit enhanced ethanol self-administration and greater hormonal and behavioral responsiveness to stress in adulthood. Whether the effects of MS are immediately evident in infancy or whether they appear only later on development is still an unanswered question This study tested sensitivity to ethanol's behavioral stimulating effects in infant rats that experienced MS from postnatal Day 1-14. MS infants exhibited significantly greater reactivity to the motor stimulating effects of 1.25 g/kg ethanol than control animals, yet greater motor suppression after 2.5 g/kg ethanol. Baseline level of response to novelty was altered in MS infants, in a nor-binaltorphimine insensitive manner, that is, despite modified activity of the kappa-opioid system. These results indicate that the consequences of chronic maternal isolation emerge early in ontogeny, affecting ethanol sensitivity in infancy.

Prenatal ethanol exposure increases ethanol intake and reduces c-Fos expression in infralimbic cortex of adolescent rats.

Prenatal ethanol exposure significantly increases later predisposition for alcohol intake, but the mechanisms associated with this phenomenon remain hypothetical. This study analyzed (Experiment 1) ethanol intake in adolescent inbred WKAH/Hok Wistar rats prenatally exposed to ethanol (2.0g/kg) or vehicle, on gestational days 17-20. Subsequent Experiments (2, 3 and 4) tested several variables likely to underlie the effect of gestational ethanol on adolescent ethanol preference, including ethanol-induced locomotor activation (LMA), ethanol-induced emission of ultrasonic vocalizations (USVs) after exposure to a rough exteroceptive stimulus, and induction of the immediate early gene C-fos in brain areas associated with processing of reward stimuli and with the retrieval and extinction of associative learning. Prenatal ethanol induced a two-fold increase in ethanol intake. Adolescents exhibited significant ethanol-induced LMA, emitted more aversive than appetitive USVs, and postnatal ethanol administration significantly exacerbated the emission of USVs. These effects, however, were not affected by prenatal ethanol. Adolescents prenatally exposed to ethanol as fetuses exhibited reduced neural activity in infralimbic cortex (but not in prelimbic cortex or nucleus accumbens core or shell), an area that has been implicated in the extinction of drug-mediated associative memories. Ethanol metabolism was not affected by prenatal ethanol. Late gestational exposure to ethanol significantly heightened drinking in the adolescent offspring of an inbred rat strain. Ethanol-induced LMA and USVs were not associated with differential ethanol intake due to prenatal ethanol exposure. Prenatal ethanol, however, altered basal neural activity in the infralimbic prefrontal cortex. Future studies should analyze the functionality of medial prefrontal cortex after prenatal ethanol and its potential association with predisposition for heightened ethanol intake.