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Chronic alcohol exposure affects the central nervous system, influences behavior, and induces neuroadaptive changes in vertebrate species including our own. The molecular mechanisms responsible for chronic alcohol effects have not been fully elucidated due to the complexity of alcohol's actions. Here we use zebrafish, a novel tool in alcohol research, to reveal a large number of genes that respond to chronic alcohol treatment. We demonstrate differential gene expression in response to chronic alcohol treatment using full genome DNA microarrays and find a total of 1914 genes to show a minimum of 2-fold and significant expression level change (1127 were up- and 787 were down-regulated). Approximately two-thirds of these genes had no known previous functional annotation. The results of the microarray analyses correlated well with those obtained on a selected subset of genes analyzed by quantitative real-time RT-PCR. Analyses of the differentially expressed genes with known annotations were enriched for a variety of molecular functions. Only a fraction of these known genes has been reported in the literature to be alcohol related. We conclude that the zebrafish is an excellent tool for the analysis of genes associated with alcohol's actions in vertebrates, one which may facilitate the discovery and better understanding of the mechanisms of alcohol abuse.

This study investigated the effects of exposure to ethanol during the brain growth spurt on a visual-discrimination (VD) and a place-learning task (PL) using intra-maze cues in the water maze. Artificially reared male Long-Evans rats were exposed to ethanol (ET) in a binge pattern from postnatal days 6-9 (6.5 g kg(-1) x day(-1); BAC approximately 330 mg/dl) or an isocaloric maltose-dextrin solution (gastrostomy control). A third suckled control group was reared by lactating dams. In experiment 1, rats were trained to discriminate horizontal- (H) versus vertical-striped (V) cues, with the positive cue providing escape from water. Groups did not differ with V+, but ET rats made more errors with H+. In experiment 2, the ET group was impaired in learning the spatial location of a submerged platform relative to intra-maze cues. In both tasks, acquisition deficits among ET rats were characterized by impairment emerging at trial 2, with intact reference memory on trial 1, and the ET group reached a comparable level of performance to controls by the end of training. In summary, because impairment was related to task characteristics, a clear distinction between impaired spatial- versus cue-based learning was not supported. However, these findings do support an effect of exposure to ethanol during the brain growth spurt on recent event, but not reference, memory.

Repeated exposure to ethanol in mice induces behavioural sensitization, a progressive increase in locomotor activity that is common to drugs of abuse. Not all mice however show sensitization to ethanol. The goal of the present study was to examine whether variability in the sensitization response to ethanol (EtOH) is differentially associated with regional brain changes in specific molecular markers associated with neuroplasticity, namely BDNF and its receptor trkB, and levels of phosphorylated cyclic AMP-regulated element-binding protein (pCREB), 14 days after withdrawal from chronic, intermittent EtOH exposure. Male DBA/2NCrl mice received 7 biweekly EtOH (2.2g/kg, i.p.) or saline (SAL) injections and were classified as Sensitized or Non-sensitized on the basis of final locomotor activity (LMA) scores. Brains were removed two weeks later for immunohistochemical and in situ hybridization analyses. Compared to SAL-treated and Non-sensitized mice, Sensitized animals showed a higher number of pCREB-immunoreactive cells in the nucleus accumbens shell (+68% and +50%, respectively) and in the bed nucleus of the stria terminalis (+61% and 46%, respectively), whereas SAL and Non-sensitized groups did not differ from each other. A different pattern was seen when BDNF and trkB mRNA levels were analyzed in the same groups. Non-sensitized mice displayed lower BDNF mRNA in several brain areas and significantly lower trkB levels throughout the brain when compared to either the Sensitized or to SAL groups, which did not differ from each other. These results indicate that sensitization to EtOH is differentially associated with increased pCREB levels in specific brain areas. The observed decrease in BDNF and trkB mRNA in the Non-sensitized group suggests the possibility that EtOH may have neurotoxic effects in a subpopulation of mice, which might in turn prevent the development of behavioural sensitization. The lack of a difference in BDNF and trkB mRNA expression between Sensitized and SAL mice suggests that EtOH sensitization may be mediated by mechanisms different from those mediating sensitization to other psychostimulants.

Zebrafish is gaining popularity in behavioral neuroscience in general and in alcohol research in particular. Alcohol is known to affect numerous molecular mechanisms depending on dose and administration regimen. Prominent among these mechanisms are several neurotransmitter systems. Here we analyze the responses of the dopaminergic and serotoninergic neurotransmitter systems of zebrafish to acute alcohol treatment (1 h long exposure of adult fish to 0.00%, 0.25%, 0.50%, or 1.00% ethyl alcohol) by testing the concentration of dopamine, its metabolite DOPAC, and serotonin and its metabolite 5-HIAA from whole brain extracts. We utilize a sensitive HPLC method and describe significant alcohol induced changes in zebrafish for the first time. We show that dopamine significantly increased in a quasi-linear dose dependent manner, DOPAC showed a smaller apparent increase which was non-significant, while both serotonin and 5-HIAA showed a significant increase only in the highest acute dose group. We discuss the methodological novelty of our work and theorize about the implications of the neurotransmitter level changes from a behavioral perspective.

Stress is often considered an important factor in the development of alcohol addiction. In rodents, various types of stressors have been shown to potentiate the effects of alcohol on behavioral responses, and to increase consumption of this substance. However, few have investigated the interaction between stress and alcohol in zebrafish. In the current study we present a repeated handling stress paradigm we developed for zebrafish, and examine whether stress alters alcohol induced behavioral and neurochemical responses. Our results show that repeated handling of zebrafish conducted for 2 consecutive days is sufficient to increase anxiety-like behavioral responses quantified 24h post-stressor. Repeatedly handled zebrafish also exhibited a reduction in the levels of serotonin's metabolite, 5-hydroxyindole acetic acid (quantified by high precision liquid chromatography) compared to unhandled controls. A 60-min acute exposure to 1% ethanol was found to significantly increase locomotor activity and decrease anxiety-like behavioral responses in stressed zebrafish but not in controls. Furthermore, unhandled control zebrafish exhibited a significant increase in whole-brain dopamine levels following exposure to ethanol but the increase was not observed in repeatedly handled fish. Our findings suggest that ethanol induced locomotor activity and anxiolysis is potentiated by handling stress and may be partially mediated by changes in dopaminergic and serotonergic activity. Overall, we demonstrate the validity of our repeated handling stressor paradigm for zebrafish, which can be used to investigate the interaction between stress and ethanol.

Chronic prenatal ethanol exposure (CPEE) can injure the developing brain, and may lead to the fetal alcohol syndrome (FAS). Previous studies have demonstrated that CPEE upregulates gamma-aminobutyric acid type A (GABA(A)) receptor expression in the cerebral cortex, and decreases functional synaptic plasticity in the hippocampus, in the adult guinea pig. This study tested the hypothesis that CPEE increases GABA(A) receptor expression in the hippocampus of guinea pig offspring that exhibit cognitive deficits in a hippocampal-dependent spatial learning task. Timed, pregnant guinea pigs were treated with ethanol (4 g/kg maternal body weight per day), isocaloric-sucrose/pair-feeding, or water throughout gestation. GABA(A) receptor subunit protein expression in the hippocampus was measured at two development ages: near-term fetus and young adult. In young adult guinea pig offspring, CPEE increased spontaneous locomotor activity in the open-field and impaired task acquisition in the Morris water maze. CPEE did not change GABA(A) receptor subunit protein expression in the near-term fetal hippocampus, but increased expression of the beta2/3-subunit of the GABA(A) receptor in the hippocampus of young adult offspring. CPEE did not change either [(3)H]flunitrazepam binding or GABA potentiation of [(3)H]flunitrazepam binding, but decreased the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding, to hippocampal GABA(A) receptors in adult offspring. Correlational analysis revealed a relationship between increased spontaneous locomotor activity and growth restriction in the hippocampus induced by CPEE. Similarly, an inverse relationship was found between performance in the water maze and the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding in the hippocampus. These data suggest that alterations in hippocampal GABA(A) receptor expression and pharmacological properties contribute to hippocampal-related behavioral and cognitive deficits associated with CPEE.

Low doses of alcohol impair movement and reduce anxiety. Most assessments of movement under ethyl alcohol (alcohol) in the rat have been tests of whole body movements, however. There has been no examination of the effects of alcohol on skilled limb movements, such as reaching for food with a forelimb. This was the purpose of the present study. Rats were trained to reach through a slot of a box with a forelimb in order to obtain a food pellet located on an external shelf. Once asymptotic performance was achieved, rats were given alcohol (20 ml of 8, 12 or 20% (v/v) solution) in separate tests to establish a relationship between alcohol ingestion and skilled reaching performance. Acute treatment with all doses of alcohol impaired postural support, but doses of 8 and 12% alcohol improved skilled reaching success. Qualitative analysis of the movements used for reaching at doses of 8 and 12% indicated that some limb components of the reaching movement were also impaired, perhaps secondarily due to impaired posture. In contrast, the reaching success of rats with unilateral dopamine depletion, induced with the neurotoxin 6-hydroxydopamine (6-OHDA) in the nigrostriatal bundle, was impaired by the same dose of alcohol that improved reaching success in control rats. The finding of improved success in reaching associated with reduced postural support in normal rats suggests a differential action of alcohol on movement subsystems underlying posture relative to skilled movement that depends upon an intact dopaminergic system. The results are also discussed with respect to the relationship of subsystems of movement and anxiety.

Repeated administration of ethanol (EtOH) in mice leads to behavioural sensitization, a progressive increase in locomotor activity. Since not all mice sensitize equally to EtOH, the objective of the present study was to determine whether variability in this response is associated with altered subunit gene expression of the N-methyl-d-aspartate receptor (NMDAR), a primary target of EtOH. We examined NR1, NR2A, and NR2B expression throughout the brain during the development phase of EtOH sensitization, as well as after a 14 day withdrawal period. Male DBA/2J mice received 5-6 injections of EtOH (2.2g/kg, i.p.) or saline (SAL) and were categorized as high- (HS) or low-sensitized (LS) on the basis of locomotor activity scores after the final injection. NMDAR subunits were analyzed by in situ hybridization in brains removed either immediately following the final EtOH injection or 14 days thereafter. At the end of development phase, LS mice showed increased NR2A expression in several brain areas compared to saline controls. LS animals also had greater NR1 expression in the nucleus accumbens core (+11%, p=0.05) and shell (+14%, p=0.04) compared to HS mice, and increased NR2B expression in hippocampal CA1 (+20%, p=0.05) relative to saline-treated animals. High-sensitized mice showed increased NR2A expression in the bed nucleus of the stria terminalis when compared to controls (+54%, p=0.02). No differences in gene expression between the treatment groups were seen 14 days after the final injection. These findings suggest that region-specific NMDAR subunits may play an important role in the variability associated with the induction of EtOH sensitization. Low-sensitized mice may be more sensitive to the NMDAR inhibitory effects of EtOH, with the NR1 and NR2A subunits potentially playing a key role in the failure to sensitize upon repeated EtOH exposure.

The zebrafish is emerging as a popular animal model for alcohol (ethanol or EtOH) addiction due to its simplicity and practical advantages. Two phenomena associated with ethanol addiction are the development of tolerance and withdrawal. Using a multi-level approach in the current study, we characterise ethanol tolerance and withdrawal in zebrafish. We first investigate the temporal trajectory of ethanol concentration in the zebrafish brain in response to an acute exposure and during withdrawal. We report that ethanol concentrations approach a steady state within 60 min of exposure to 0.50% and 1.00% v/v ethanol and rapidly decline and return to zero within 60 min following withdrawal from chronic ethanol exposure (0.50% v/v). We characterise the changes associated with ethanol tolerance and withdrawal in zebrafish by focusing on three domains relevant to ethanol addiction: motor patterns, physiological responses (i.e. cortisol levels), and neurochemical alterations. The use of multiple domains of investigation allowed an in-depth analysis of ethanol induced changes in zebrafish.