Pharmacological targeting of the GABAʙ receptor alters Drosophila's behavioural responses to alcohol

When exposed to ethanol, Drosophila melanogaster display a variety of addiction‐like behaviours similar to those observed in mammals. Sensitivity to ethanol can be quantified by measuring the time at which 50% of the flies are sedated by ethanol exposure (ST50); an increase of ST50 following multiple ethanol exposures is widely interpreted as development of tolerance to ethanol. Sensitivity and tolerance to ethanol were measured after administration of the gamma‐aminobutyric acid receptor B (GABAʙ) agonist (SKF 97541) and antagonist (CGP 54626), when compared with flies treated with ethanol alone. Dose‐dependent increases and decreases in sensitivity to ethanol were observed for both the agonist and antagonist respectively. Tolerance was recorded in the presence of GABAʙ drugs, but the rate of tolerance development was increased by SKF 97451 and unaltered in presence of CGP 54626. This indicates that the GABAʙ receptor contributes to both the sensitivity to ethanol and mechanisms by which tolerance develops. The data also reinforce the usefulness of Drosophila as a model for identifying the molecular components of addictive behaviours and for testing drugs that could potentially be used for the treatment of alcohol use disorder (AUD)

The Role of G Protein In Alcohol Related Behaviours Using Drosophila melanogaster As A Model

Ethanol, classified as a drug, affects the central nervous system, and its consumption has been linked to the development of several behaviours including tolerance and dependence. Alcohol tolerance is defined as the need for higher doses of alcohol to induce the same changes observed in the initial exposure or where repetitive exposures of the same alcohol dose induce a lower response. Ethanol has been shown to interact with numerous targets and ultimately influence both short and long term adaptation at the cellular and molecular level in brain [1]. These adaptation processes are likely to involve signalling molecules: our work has focussed on G proteins gene expression. Using both wild type and several mutant fruit fly (Drosophila melanogaster) as a model for behaviour and molecular studies, we observed significant increases in sedation time (ST50) in response to alcohol (P<0.001) Fig.A. We also observed a consistent and significant decrease of Gq protein mRNA expression in Drosophila dUNC and DopR2 mutants chronically exposed to alcohol (*P<0.05). Fig B. Method: Six male flies were observed in drosophila polystyrene 25 x 95mm transparent vial in between cotton plugs. To the top plug, 500uL of 100% ethanol was added. Time till 50% of the flies were sedated was recorded on each day following the schedule. Fig. C (n=4-6). Using RT-PCR, we also quantified G protein mRNA expression levels one hour post initial 30 minutes of ethanol expression on day 1 and day 3 relative to expression in naïve flies.(n=2) [A] Increase in sedation time indicative of tolerance in different mutant lines and wild type flies. Six male flies were used in each experiment and (n= 4-6. ***P<0.001 unpaired t tests). [B] RT-PCR results showing significant reduction in Gq mRNA in flies chronically exposed to alcohol. (n=2. *P<0.05) [C] Alcohol exposure schedule. (1) Kaun K.R., R. Azanchi, Z. Maung, J. Hirsh, U. Heberlein. (2011). A Drosophila model for alcohol reward. Nature Neuroscience. 14 (5), 612–619

Ethanol-induced G-protein subunit expression changes in D2 receptor deficient Drosophila

Alcohol abuse and addiction impact on users’ quality of life and have substantial implications for health services. Understanding the mechanisms by which alcohol modifies cellular and molecular mechanisms associated with chronic abuse, could lead to improved pharmaceutical interventions to overcome alcohol addiction. Alcohol acts through multiple receptor systems and, like other addictive drugs, causes prolonged or permanent changes in gene expression. Dopamine release and changes in gene expression of elements of the cAMP-CREB-DeltaFosB pathways have been associated to addictive behaviours. However, the mechanisms linking ethanol with long-term changes in the reward pathways are not fully understood. In this work, we have focused on measuring changes in G-protein gene expression in a Drosophila melanogaster ethanol tolerance model. Exposure of Drosophila to ethanol vapour causes sedation in the flies, but multiple exposure increases the sedation time, which is considered a manifestation of ethanol tolerance. Using quantitative real-time polymerase chain reaction (qRT-PCR), we have measured G-protein mRNA in flies that have experienced zero, one or three ethanol exposures at 24 hours intervals. When measured in a wild type population, changes in G-protein levels were variable. However in a sub-population of Drosophila that we selected for high ethanol sensitivity we observed a non-statistically significant decrease of two Gα-protein subunits: Gi and Gq. These same changes were observed at a statistically significant level in two Drosophila mutant lines characterised by a deletion of Dopamine D2 receptor and a non-functional of Gq subunit respectively. These two Drosophila lines also displayed an altered sensitivity to ethanol while retaining the tolerance response to alcohol. These data indicate that when measured in genetically homogeneous populations ethanol induced G-proteins gene expression changes can be detected, but the persistence of this effect in flies lacking D2 receptors suggests that these G-proteins subunits changes do not utilise the previously described D2 receptor dependant mechanisms associated with addictive drugs

Selective 5HT3 antagonists and sensory processing: A systematic review

Ondansetron is a selective serotonin (5HT3) receptor antagonist that is under evaluation as an adjunctive treatment for schizophrenia, and a novel treatment for hallucinations in Parkinson’s disease. Ondansetron reverses sensory gating deficits and improves visuoperceptual processing in animal models of psychosis, but it is unclear to what extent preclinical findings have been replicated in humans. We systematically reviewed human studies that evaluated the effects of ondansetron and other 5HT3 receptor antagonists on sensory gating deficits or sensory processing. Of 11 eligible studies, eight included patients with schizophrenia who were chronically stable on antipsychotic medication; five measured sensory gating using the P50 suppression response to a repeated auditory stimulus; others included tests of visuoperceptual function. Three studies in healthy participants included tests of visuoperceptual and sensorimotor function. A consistent and robust finding (five studies) was that ondansetron and tropisetron (5HT3 antagonist and α7-nicotinic receptor partial agonist) improved sensory gating in patients with schizophrenia. Tropisetron also improved sustained visual attention in non-smoking patients. There was inconsistent evidence of the effects of 5HT3 antagonists on other measures of sensory processing, but interpretation was limited by the small number of studies, methodological heterogeneity and the potential confounding effects of concomitant medication in patients. Despite these limitations, we found strong evidence that selective 5HT3 antagonists (with or without direct α7-nicotinic partial agonist effects) improved sensory gating. Future studies should investigate how this relates to potential improvement in neurocognitive symptoms in antipsychotic naive patients with prodromal or milder symptoms, in order to understand the clinical implications

G-protein αq gene expression plays a role in alcohol tolerance in Drosophila melanogaster

Ethanol is a psychoactive substance causing both short- and long-term behavioural changes in humans and animal models. We have used the fruit fly Drosophila melanogaster to investigate the effect of ethanol exposure on the expression of the Gαq protein subunit. Repetitive exposure to ethanol causes a reduction in sensitivity (tolerance) to ethanol, which we have measured as the time for 50% of a set of flies to become sedated after exposure to ethanol (ST50). We demonstrate that the same treatment that induces an increase in ST50 over consecutive days (tolerance) also causes a decrease in Gαq protein subunit expression at both the messenger RNA and protein level. To identify whether there may be a causal relationship between these two outcomes, we have developed strains of flies in which Gαq messenger RNA expression is suppressed in a time- and tissue-specific manner. In these flies, the sensitivity to ethanol and the development of tolerance are altered. This work further supports the value of Drosophila as a model to dissect the molecular mechanisms of the behavioural response to alcohol and identifies G proteins as potentially important regulatory targets for alcohol use disorders

Pharmacological Modulation of Alcohol Tolerance via GABA-B receptors in Drosophila melanogaster

Aims: Ethanol induced behaviours are mediated via the GABA-B receptor, but mechanisms remain unclear. Drosophila melanogaster respond to alcohol by developing addiction-like behaviours such as changes to ethanol sensitivity (tolerance). The aim of this study was to determine the effect of GABA-B receptor drugs on the development of alcohol tolerance in male adult Drosophila to further elucidate the role of GABA-B receptors. Methods: The GABA-B receptor agonist (SKF-97451) and antagonist (CGP-54626) were administered orally for 24h before a three-day assay to challenge ethanol tolerance in wild type and GABA-B receptor mutant flies. ST50, defined as 50% of flies sedated from ethanol exposure, was used as a measure of sensitivity on each day. Results are n=6 with 10 flies per assay. Results: Both agonist and antagonist increased the ST50 in WT and GABA-B R1 KO when compared to control. Notably, the GABA-B R2 KO did not respond to either agonist or antagonist and differed significantly from the R1 KO and WT response suggesting a complex mechanism of action. Conclusion: The data provides evidence that the GABA-B receptors are involved in modulating the process of ethanol tolerance in Drosophila melanogaster. Further work with different mutant flies and drug delivery protocols will probe the role of these receptors in alcohol addiction