Ethanol-induced changes in G-proteins gene expression

The cellular and molecular mechanism underlying addictive behaviours in humans is still not fully understood. Several studies have indicated that the expression of certain genes in the reward pathways of the central nervous system are altered following repeated exposure to addictive drugs. Our studies are focussed on ethanol and on its effect on the expression of G proteins, which are associated with several different neurotransmitter receptors. In our study we use the fruit fly Drosophila Melanogaster as a model organism. This insect displays classical tolerance behaviour when exposed to ethanol and contains the same receptors systems found in mammalian reward pathways. Our findings indicate that the expression of some of the G protein subtypes is altered following acute and chronic ethanol exposure. We have also observed that there is considerable variation among the wild type population in the response to alcohol and we are carrying out parallel studies to isolate strains of flies with different response behaviour. The work is aimed at better understanding the role of G proteins in the signalling response to ethanol in the fly. The information obtained should also further elucidate addiction mechanism in mammalian systems. This work is part of the internal collaboration of UEL’s Substance Use and Misuse (SUM) network

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

Amphetamine and pseudoephedrine cross-tolerance measured by c-Fos protein expression in brains of chronically treated rats

Background: Pseudoephedrine is a drug commonly prescribed as a nasal decongestant and bronchodilator and is also freely available in cold remedies and medications. The structural and pharmacological similarity of pseudoephedrine to amphetamine has led to evaluation of its psychomotor stimulant properties within the central nervous system. Previous investigations have shown that the acute responses to pseudoephedrine were similar to those of amphetamine and other psychostimulants. Results: This study examined the effect of chronic administration of pseudoephedrine in rat nucleus accumbens and striatum and identified three further similarities to amphetamine. (i) Chronic exposure to pseudoephedrine reduced the c-Fos response to acute pseudoephedrine treatment suggesting that pseudoephedrine induced tolerance in the animals. (ii) In animals chronically treated with amphetamine or pseudoephedrine the acute c-Fos response to pseudoephedrine and amphetamine was reduced respectively as compared to naïve animals indicating cross-tolerance for the two drugs. (iii)The known involvement of the dopamine system in the response to amphetamine and pseudoephedrine was further confirmed in this study by demonstrating that pseudoephedrine similarly to amphetamine, but with lower potency, inhibited [3H]dopamine uptake in synaptosomal preparations. Conclusion: This work has demonstrated further similarities of the effect of pseudoephedrine to those of amphetamine in brain areas known to be associated with drug addiction. The most significant result presented here is the cross tolerance effect of amphetamine and psudoephedrine. This suggests that both drugs induce similar mechanisms of action in the brain. Further studies are required to establish whether despite its considerable lower potency, pseudoephedrine could pose health and addiction risks in humans similar to that of known psychostimulants

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

Jigsaw Recovery: The Spatio-temporalities of Alcohol Abuse and Recovery in a Non-interventionist, Peer-led Service

Research highlights that recovery from substance use is a process facilitated by relational factors, resources and therapeutic practices embedded in places conducive to recovery. However, the accessibility of such resources for those with complex needs, and the therapeutic potential of peer-led spaces needs contextualizing in both time and place. We examined the characteristics of a social space employing a noninterventionist peer-led approach for active alcohol users. Individuals prioritized the management of everyday life over recovery, especially abstinence. This space acted as a replacement “jigsaw”; interrupting the temporal, spatial and social aspects of active use. Flexible approaches allowing choice in recovery pathways appear significant for this population

β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

Background The mammalian inner ear contains the organ of Corti which is responsible for the conversion of sound into neuronal signals. This specialised epithelial tissue is the product of a complex developmental process where a common precursor cell type differentiates into the sound transducing hair cells and the non-innervated supporting cells. We hypothesised that integrin proteins, which are involved in cell attachment to extracellular matrix proteins and cellular signalling, play a role in the differentiation process of the precursor inner ear epithelial cells. To test our hypothesis we have utilised a cell line (OC-2) derived from E13 embryonic immortomouse inner ears. In vitro, by switching the incubation temperature from 33°C to 39°C, the OC-2 cells can be induced to differentiate and express hair cells markers, such as Myosin VIIa. The OC-2 cells are thus a useful model system for testing mechanism of hair cells differentiation. Results We have identified 4 integrin subunits which are expressed in OC-2 cells: α6, αv, β1 and β3. Among these, the relative level of expression of the αv, β1 and β3 subunits increased in a time dependent manner when the cells were exposed to the differentiating temperature of 39°C, most notably so for β3 which was not detectable at 33°C. Treatment of fully differentiated OC-2 cells with siRNA against the four integrin subunits reduced the expression of not only the respective integrin proteins but also of the hair cell marker Myosin VIIa. Conversely over-expression of β3 was sufficient to induce the expression of Myosin VIIa at 33°C. Conclusions Our data demonstrate that modulation of integrin expression is associated with the differentiation process of the OC-2 cells. This suggests that the maturation of the organ of Corti, from where OC-2 cells are derived, may also depend on changes of gene expression associated with integrin expression

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

Role of gamma-aminobutyric acid-B receptors in ethanol induced behaviours

Alcohol is one of the most widely used and socially accepted psychoactive substances in the world, and its misuse was accountable for 3.3 million alcohol related deaths in the world in 2015. Whilst it is known that ethanol enhances the actions of the GABA-B receptor, the role of the stimulation of this receptor in inducing acute and chronic effects, remains to be investigated and identified. The fruit fly, Drosophila melanogaster, offers the possibility to investigate behaviours such as preference and tolerance to alcohol, and to challenge them with pharmacological agents. In this study, the GABA-B receptor agonist (SKF 97451) and antagonist (CGP 54626) were used to challenge the development of tolerance and the onset of preference to alcohol in wild type flies and in mutant lines with putative disruptions of GABAB receptor 1 or 2 subunit genes. The results indicate that the GABA-B receptors are indeed part of a complex mechanism that result in alcohol induced behavioural changes. The data support the usefulness of the Drosophila model and the need for further investigations