RESUMEN

Introduction: Mechanisms that dictate the preference for ethanol and its addiction are not only restricted to the central nervous system (CNS). An increasing body of evidence has suggested that abusive ethanol consumption directly affects the immune system, which in turn interacts with the CNS, triggering neuronal responses and changes, resulting in dependence on the drug. It is known that neuroinflammation and greater immune system reactivity are observed in behavioral disorders and that these can regulate gene transcription. However, there is little information about these findings of the transcriptional profile of reward system genes in high consumption and alcohol preference. In this regard, there is a belief that, in the striatum, an integrating region of the brain reward system, the interaction of the immune response and the transcriptional profile of the Lrrk2 gene that is associated with loss of control and addiction to ethanol may influence the alcohol consumption and preference. Given this information, this study aimed to assess whether problematic alcohol consumption affects the transcriptional profile of the Lrrk2 gene, neuroinflammation, and behavior and whether these changes are interconnected. Methods: An animal model developed by our research group has been used in which male C57BL/6 mice and knockouts for the Il6 and Nfat genes were subjected to a protocol of high fat and sugar diet intake and free choice of ethanol in the following stages: Stage 1 (T1)-Dietary treatment, for 8 weeks, in which the animals receive high-calorie diet, High Sugar and Butter (HSB group), or standard diet, American Institute of Nutrition 93-Growth (AIN93G group); and Stage 2 (T2)-Ethanol consumption, in which the animals are submitted, for 4 weeks, to alcohol within the free choice paradigm, being each of them divided into 10 groups, four groups continued with the same diet and in the other six the HSB diet is substituted by the AIN93G diet. Five groups had access to only water, while the five others had a free choice between water and a 10% ethanol solution. The weight of the animals was evaluated weekly and the consumption of water and ethanol daily. At the end of the 12-week experiment, anxiety-like behavior was evaluated by the light/dark box test; compulsive-like behavior by Marble burying, transcriptional regulation of genes Lrrk2, Tlr4, Nfat, Drd1, Drd2, Il6, Il1ß, Il10, and iNOS by RT-qPCR; and inflammatory markers by flow cytometry. Animals that the diet was replaced had an ethanol high preference and consumption. Results and discussion: We observed that high consumption and preference for ethanol resulted in (1) elevation of inflammatory cells in the brain, (2) upregulation of genes associated with cytokines (Il6 and Il1ß) and pro-inflammatory signals (iNOS and Nfat), downregulation of anti-inflammatory cytokine (Il10), dopamine receptor (Drd2), and the Lrrk2 gene in the striatum, and (3) behavioral changes such as decreased anxiety-like behavior, and increased compulsive-like behavior. Our findings suggest that interactions between the immune system, behavior, and transcriptional profile of the Lrrk2 gene influence the ethanol preferential and abusive consumption.

RESUMEN

BACKGROUND: The motivations for and effects of ethanol consumption vary considerably among individuals, and as such, a significant proportion of the population is prone to substance abuse and its negative consequences in the physical, social, and psychological spheres. In a biological context, the characterization of these phenotypes provides clues for understanding the neurological complexity associated with ethanol abuse behavior. Therefore, the objective of this research was to characterize four ethanol preference phenotypes described in zebrafish: Light, Heavy, Inflexible, and Negative Reinforcement. METHODS: To do this, we evaluated the telomere length, mtDNA copy number using real-time quantitative PCR (qPCR), and the activity of these antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the brain, and the interactions between these biomarkers. Changes observed in these parameters were associated with ethanol consumption and alcohol abuse. RESULTS: The Heavy, Inflexible, and Negative Reinforcement phenotypes showed ethanol preference. This was particularly the case with the Inflexible phenotype, which was the group with the greatest ethanol preference. These three phenotypes showed telomere shortening as well as high SOD/CAT and/or GPx activities, while the Heavy phenotype also showed an increase in the mtDNA copy number. However, the Light phenotype, containing individuals without ethanol preference, did not demonstrate any changes in the analyzed parameters even after being exposed to the drug. Additionally, the PCA analysis showed a tendency to cluster the Light and Control groups differently from the other ethanol preference phenotypes. There was also a negative correlation between the results of the relative telomere length and SOD and CAT activity, providing further evidence of the biological relationship between these parameters. CONCLUSIONS: Our results showed differential molecular and biochemistry patterns in individuals with ethanol preference, suggesting that the molecular and biochemical basis of alcohol abuse behavior extends beyond its harmful physiological effects, but rather is correlated with preference phenotypes.

Asunto(s)

RESUMEN

Alcohol consumption is associated with alterations in memory and learning processes in humans and animals. In this context, research models such as the zebrafish (Danio rerio) arise as key organisms in behavioral and molecular studies that attempt to clarify alterations in the Central Nervous System (CNS), like those related to alcohol use. Accordingly, we used the zebrafish as a model to evaluate the effects of ethanol on the learning and memory process, as well as its relationship with behavior and transcriptional regulation of lrfn2, lrrk2, grin1a, and bdnf genes in the brain. To this end, for the memory and learning evaluation, we conducted the Novel Object Recognition test (NOR); for behavior, the Novel Tank test; and for gene transcription, qPCR, after 2 h, 24 h, and 8 days of ethanol exposure. As a result, we noticed in the NOR that after 8 days of ethanol exposure, the control group spent more time exploring the novel object than when compared to 2 h post-exposure, indicating that naturally zebrafish remember familiar objects. In animals in the Treatment group, however, no object recognition behavior was observed, suggesting that alcohol affected the learning and memory processes of the animals and stimulated an anxiolytic effect in them. Regarding transcriptional regulation, 24 h after alcohol exposure, we found hyper-regulation of bdnf and, after 8 days, a hypo-regulation of lrfn2 and lrrk2. To conclude, we demonstrated that ethanol exposure may have influenced learning ability and memory formation in zebrafish, as well as behavior and regulation of gene transcription. These data are relevant for further understanding the application of zebrafish in research associated with ethanol consumption and behavior.

Asunto(s)

RESUMEN

Background: Genetics influence the vulnerability to alcohol use disorders, and among the implicated genes, three previous studies have provided evidences for the involvement of LRRK2 in alcohol dependence (AD). LRRK2 expression is broadly dysregulated in postmortem brain from AD humans, as well as in the brain of mice with alcohol dependent-like behaviors and in a zebrafish model of alcohol preference. The aim of the present study was to evaluate the association of variants in the LRRK2 gene with AD in multiethnic populations from South and North America. Methods: Alcohol-screening questionnaires [such as CAGE and Alcohol Use Disorders Identification Test (AUDIT)] were used to determine individual risk of AD. Multivariate logistic regression analyses were done in three independent populations (898 individuals from Bambuí, Brazil; 3,015 individuals from Pelotas, Brazil; and 1,316 from the United States). Linkage disequilibrium and conditional analyses, as well as in silico functional analyses, were also conducted. Results: Four LRRK2 variants were significantly associated with AD in our discovery cohort (Bambuí): rs4768231, rs4767971, rs7307310, and rs1465527. Two of these variants (rs4768231 and rs4767971) were replicated in both Pelotas and US cohorts. The consistent association signal (at the LRRK2 locus) found in populations with different genetic backgrounds reinforces the relevance of our findings. Conclusion: Taken together, these results support the notion that genetic variants in the LRRK2 locus are risk factors for AD in humans.

RESUMEN

Wistar Audiogenic Rat is an epilepsy model whose animals are predisposed to develop seizures induced by acoustic stimulation. This model was developed by selective reproduction and presents a consistent genetic profile due to the several generations of inbreeding. In this study, we performed an analysis of WAR RNA-Seq data, aiming identified at genetic variants that may be involved in the epileptic phenotype. Seventeen thousand eighty-five predicted variants were identified as unique to the WAR model, of which 15,915 variants are SNPs and 1,170 INDELs. We filter the predicted variants by pre-established criteria and selected five for validation by Sanger sequencing. The genetic variant c.14198T>C in the Vlgr1 gene was confirmed in the WAR model. Vlgr1 encodes an adhesion receptor that is involved in the myelination process, in the development of stereocilia of the inner ear, and was already associated with the audiogenic seizures presented by the mice Frings. The transcriptional quantification of Vlgr1 revealed the downregulation this gene in the corpus quadrigeminum of WAR, and the protein modeling predicted that the mutated residue alters the structure of a domain of the VLGR1 receptor. We believe that Vlgr1 gene may be related to the predisposition of WAR to seizures and suggest the mutation Vlgr1/Q4695R as putative causal variant, and the first molecular marker of the WAR strain.

RESUMEN

The high prevalence of obesity and associated metabolic disorders are one of the major public health problems worldwide. Among the main causal factors of obesity, excessive consumption of food rich in sugar and fat stands out due to its high energy density. The regulation of food intake relies on hypothalamic control by the action of several neuropeptides. Excessive consumption of hypercaloric diets has impact in the behavior and in the gut microbiota. In the present study, we used a high-sugar and fat (HSB) diet for 12 weeks to induce obesity in C57BL/6 mice and to investigate its effects on the gut microbiota, hypothalamic peptides, and behavior. We hypothesize that chronic consumption of HSB diet can change the behavior. Additionally, we also hypothesize that changes in gut microbiota can be associated with changes in the transcriptional regulation of hypothalamic peptides and behavior. To evaluate the gut microbiota, we performed the sequencing of 16S rRNA gene, which demonstrate that HSB diet modulates the gut microbiota with an increase in the Firmicutes and Actinobacteria phylum and a decrease of Bacteroidetes phylum. The real time qPCR revealed that HSB-fed mice presented changes in the transcriptional regulation of hypothalamic neuropeptides genes such as Npy, Gal and Galr1. The Marble-burying and Light/dark box tests also showed an alteration in anxiety and impulsive behaviors for the HSB-fed mice. Our data provides evidence that obesity induced by HSB diet consumption is associated with alterations in gut microbiota and behavior, highlighting the multifactorial characteristics of this disease.

Asunto(s)

RESUMEN

Ethanol consumption is correlated with different neurobiological and behavioral impairments. Acute and chronic exposure to this drug is associated with alterations in the regulation of the mesolimbic dopaminergic system as well as with transcriptional modulation of other receptors in the central nervous system and can unleash seeking behavior or behavioral adaptations and phenotypes such as loss of control, dependence and tolerance. In the present work, we characterized the chronological effects of acute and chronic intermittent exposure to ethanol (1% v/v) in an adult zebrafish population (Danio rerio). During sixteen days of ethanol exposure, we associated the neuromodulation of target genes (drd1, drd2, gabra2a, gabbr1a, gabbr1b) in the central nervous system with behavioral parameters, assessed by social preference, antipredatory capacity and anxiety-like analysis. Transcriptional and behavioral data were collected in days 0, 1, 4, 8, 12 and 16, after ethanol exposure. In days 1 and 4, ethanol exposure increased exploratory behavior regardless of the risk involved (less time spent close to conspecifics and lower avoidance reaction to predator). Along with the reduction of drd2, grin1a and gabra2a transcription seen in the same days, these results suggest an anxiolytic effect of acute ethanol exposure. Interestingly, in days 8, 12 and 16, an attenuation of the behavioral effects was observed. The social preference, antipredatory behavior, perception and exploration parameters were reconstituted. This behavioral re-establishment, accompanied by the increase in drd1, drd2 and gabbr1a transcription in the 8th day could be an indicative of an adaptation to chronic exposure to ethanol. The modulation of drd2 gene combined with the behavioral characterization observed in the study suggests this signalling pathway as a key participant in the phenotypic outcomes of a long-term chronic exposure to ethanol. Lastly, our results reaffirm the ethanol deleterious impacts in perception, ability to respond to adverse stimuli and in anxiety-like behavior.

Asunto(s)

RESUMEN

Due to its multifactorial and yet to be fully understood origin, ethanol addiction is a field that still requires studies for the elucidation of novel genes and pathways that potentially influence the establishment and maintenance of addiction-like phenotypes. In this context, the present study aimed to evaluate the role of the LRRK2 pathway in the modulation of ethanol preference behavior in Zebrafish (Danio rerio). Using the behavioral Conditioned Place Preference (CPP) paradigm, we accessed the preference of animals for ethanol. Next, we evaluated the transcriptional regulation of the gene lrrk2 and the receptors drd1, drd2, grin1a, gria2a, and gabbr1b in the zebrafish brain. Additionally, we used a selective inhibitor of Lrrk2 (GNE-0877) to assess the role of this gene in the preference behavior. Our results revealed four distinct ethanol preference phenotypes (Light, Heavy, Negative Reinforcement, and Inflexible), each showing different transcriptional regulation patterns of the drd1, drd2, grin1a, gria2a, and gabbr1b receptors. We showed that the lrrk2 gene was hyperregulated only in the brains of the animals with the Inflexible phenotype. Most importantly, we showed, for the first time in the context of preference for ethanol, that treatment with the GNE-0877 inhibitor modulates the transcription of the target receptor genes and reduces the preference for ethanol in the animals of the Inflexible group. This result corroborates the hypothesis that the LRRK2 pathway is involved in the inflexible preference for ethanol behavior. Lastly, we identified a possible pharmacological target for the treatment of abusive preference behavior for ethanol.

Asunto(s)

RESUMEN

The bidirectional and positive relation between the ingestion of fat and alcohol has become the subject of extensive discussion. However, this relation is more studied in animal models of binge eating with intermittent access of high-fat diet or in a model of short period of this diet consumption. Here, we developed a model to elucidate how chronic high-fat diet and its withdrawal influence alcohol intake (two-bottle choice) and anxiety behavior (marble burying test). In the first experimental stage, animals were fed on standard (AIN93G) or high sugar and butter (HSB) diet for 8 weeks. Then, a protocol of free-choice between water and a 10% alcohol solution was introduced, and the HSB diet was replaced with AIN93G in two experimental groups. The result obtained with this model point out that the relation among high-fat diet consumption and alcohol intake appears to depend on the presence or absence of the diet when alcohol intake is evaluated, and that an imbalance in the mesocorticolimbic dopaminergic pathway, observed by the transcriptional regulation of the dopamine receptors (Drd1/Drd2) and GABAB receptors subunit (Gabbr1/Gabbr2), can be driving the alcohol intake.

Asunto(s)

RESUMEN

Alcohol use disorder (AUD) is a complex multifactorial disease with heritability of ∼50% and corresponds to the state in which the body triggers a reinforcement or reward compulsive behavior due to ethanol consumption, even when faced with negative consequences. Although several studies have shown the impact of high ethanol intake on the prefrontal cortex (PFC) gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with relapsing. In this study, we used a chronic three-bottle free-choice mouse model to investigate the PFC transcriptome in three different groups of mice drinkers: 'Light drinkers' (preference for water throughout the experiment); 'Heavy drinkers' (preference for ethanol with a non-compulsive intake), and 'Inflexible drinkers' (preference for ethanol with a compulsive drinking component). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the PFC, a brain region critical for the development and maintenance of alcohol addiction. We found that the Camk2a gene showed a downregulated profile only in the Inflexible when compared to the Light drinkers group, the Camk2n1 and Pkp2 genes showed an upregulated profile only in the Inflexible drinkers when compared to the Control group, and the Gja1 gene showed an upregulated profile in the Light and Inflexible drinkers when compared to the Control group. These different transcription patterns have been associated to the presence of alcohol, in the Camk2n1 and Gja1 genes; to the amount of ethanol consumed, in the Camk2a gene; and to the loss of control in the alcohol consumption, in the Pkp2 gene. Here, we provide, for the first time, the potential involvement of the Pkp2 gene in the compulsivity and loss of control over the voluntary ethanol consumption.

Asunto(s)

RESUMEN

Alcoholism is a psychiatric disorder that composes one of the principal causes of health disabilities in the world population. Furthermore, the available pharmacotherapy is limited. Therefore, this research was carried out to better understand the basis of the underlying neurobiological processes of this disorder and to discover potential therapeutic targets. Real-time PCR analysis was performed in the amygdala nuclei region of the brain of mice exposed to a chronic three-bottle free-choice model (water, 5 and 10% v/v ethanol). Based on individual ethanol intake, the mice were classified into three groups: "compulsive-like" (i.e., ethanol intake not affected by quinine adulteration), "ethanol-preferring" and "ethanol non-preferring". A fourth group had access only to tap water (control group). The candidate gene ACSS2 was genotyped in human alcoholics by real-time polymerase chain reaction using the markers rs6088638 and rs7266550. Seven genes were picked out (Acss2, Acss3, Acat1, Acsl1, Acaa2, Hadh, and Hadhb) and the mRNA level of the Acss2 gene was increased only in the "compulsive-like" group (p = 0.004). The allele frequency of rs6088638 for the gene ACSS2 was higher in the Alcoholic human group (p = 0.03), although sample size was very small. The gene ACSS2 is associated with alcoholism, suggesting that biochemical pathways where it participates may have a role in the biological mechanisms susceptible to the ethanol effects.

Asunto(s)

RESUMEN

Alcoholism is a complex multifactorial disorder with a strong genetic influence. Although several studies have shown the impact of high ethanol intake on the striatal gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with the two major concerns in addiction: the excessive drug consumption and relapsing. In this study, we used a chronic three-bottle free-choice murine model to address striatal transcript regulation among animals with different ethanol intakes and preferences: Light Drinkers (preference for water throughout the experiment), Heavy Drinkers (preference for ethanol with a non-compulsive intake) and Inflexible Drinkers (preference for ethanol and simultaneous loss of control over the drug intake). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the striatum, a brain region critical for the development of alcohol addiction. We found that the transcripts of the Lrrk2 gene, which encodes a multifunctional protein with kinase and GTPase activities, is upregulated only in Inflexible Drinkers suggesting, for the first time, that the Lrrk2 pathway plays a major role in the compulsive ethanol intake behaviour of addicted subjects.

Asunto(s)

RESUMEN

Previous studies have suggested that γ-aminobutyric acid-B (GABA(B)) receptor agonists effectively reduce ethanol intake. The quantification using real-time polymerase chain reaction of Gabbr1 and Gabbr2 mRNA from the prefrontal cortex, hypothalamus, hippocampus, and striatum in mice exposed to an animal model of the addiction developed in our laboratory was performed to evaluate the involvement of the GABA(B) receptor in ethanol consumption. We used outbred, Swiss mice exposed to a three-bottle free-choice model (water, 5% v/v ethanol, and 10% v/v ethanol) that consisted of four phases: acquisition (AC), withdrawal (W), reexposure (RE), and quinine-adulteration (AD). Based on individual ethanol intake, the mice were classified into three groups: "addicted" (A group; preference for ethanol and persistent consumption during all phases), "heavy" (H group; preference for ethanol and a reduction in ethanol intake in the AD phase compared to AC phase), and "light" (L group; preference for water during all phases). In the prefrontal cortex in the A group, we found high Gabbr1 and Gabbr2 transcription levels, with significantly higher Gabbr1 transcription levels compared with the C (ethanol-naive control mice), L, and H groups. In the hippocampus in the A group, Gabbr2 mRNA levels were significantly lower compared with the C, L, and H groups. In the striatum, we found a significant increase in Gabbr1 transcription levels compared with the C, L, and H groups. No differences in Gabbr1 or Gabbr2 transcription levels were observed in the hypothalamus among groups. In summary, Gabbr1 and Gabbr2 transcription levels were altered in cerebral areas related to drug taking only in mice behaviorally classified as "addicted" drinkers, suggesting that these genes may contribute to high and persistent ethanol consumption.

Asunto(s)

RESUMEN

Baclofen, a GABA(B) agonist, reduces ethanol intake in animals and humans, but the contrary or no effect was also reported. Our previous study demonstrated that mice characterized as "loss of control over ethanol intake" had different Gabbr1 and Gabbr2 transcription levels, which express, respectively, the GABA(B1) and GABA(B2) subunits in brain areas related to addictive behavior. In the present study, we tested baclofen on ethanol intake in mice exposed to the free-choice paradigm. Adult male Swiss mice, individually housed, had free access to three bottles: ethanol (5% and 10%) and water. The protocol had four phases: acquisition (AC, 10 weeks), withdrawal (W, 4 cycles during 2 weeks of 2 day-free-choice and 2 day-only-water), reexposure (RE, 2 weeks), and adulteration of ethanol solutions with quinine (AD, 2 weeks). Mice characterized as "loss of control" (A, n=11, preference for ethanol in AC and maintenance of ethanol intake levels in AD), heavy (H, n=11, preference for ethanol in AC and reduction of ethanol intake levels in AD), and light (L, n=16, preference for water in all phases) drinkers were randomly distributed into two subgroups receiving either intraperitoneal injections of all doses of baclofen (1.25, 2.5, and 5.0mg/kg, given each dose twice in consecutive days) or saline, being exposed to free-choice. Fluid consumption was measured 24h later. Baclofen reduced ethanol intake in group L. In group H a reduction compared to AC was observed. Group A maintained their high ethanol intake even after baclofen treatment. Activation of the GABA(B) receptor depends on the precise balance between the GABA(B1) and GABA(B2) subunits, so the disproportionate transcription levels, we reported in group A, could explain this lack of response to baclofen. These data highlight the importance to test baclofen in individuals with different ethanol drinking profiles, including humans.

Asunto(s)