Circadian Genes Differentially Affect Tolerance to Ethanol in Drosophila

Abstract

Background There is a strong relationship between circadian rhythms and ethanol responses. Ethanol consumption has been shown to disrupt physiological and behavioral circadian rhythms in mammals (Spanagel et al., 2005b). The Drosophila central circadian pacemaker is composed of proteins encoded by the per, tim, cyc, and Clk genes. Using Drosophila mutant analysis we asked whether these central components of the circadian clock make the equivalent contribution towards ethanol tolerance and whether rhythmicity itself is necessary for tolerance. Methods We tested flies carrying mutations in core clock genes for the capacity to acquire ethanol tolerance. Tolerance was assayed by comparing the sedation curves of populations during their first and second sedation. Animals that had acquired tolerance sedated more slowly. Movement was also monitored as the flies breathe the ethanol vapor to determine if other facets of the ethanol response were affected by the mutations. Gas chromatography was used to measure internal ethanol concentration. Constant light was used to non-genetically destabilize the PER and TIM proteins. Results A group of circadian mutations, all of which eliminate circadian rhythms, do not disrupt tolerance identically. Mutations in per, tim, and cyc completely block tolerance. However, a mutation in Clk does not interfere with tolerance. Constant light also disrupts the capacity to acquire tolerance. These lines did not differ in ethanol absorption. Conclusions Mutations affecting different parts of the intracellular circadian clock can block the capacity to acquire rapid ethanol tolerance. However, the role of circadian genes in ethanol tolerance is independent of their role in producing circadian rhythmicity. The interference in the capacity to acquire ethanol tolerance by some circadian mutations is not merely a downstream effect of a nonfunctional circadian clock, instead these circadian genes play an independent role in ethanol tolerance.

The FlyBar: Administering Alcohol to Flies

“…Thus, the inebriometer may not be well suited for many undergraduate teaching laboratories with limited budgets or space, or for researchers performing circadian assays. Another method of delivering alcohol to flies and measuring sedation involves placing a small amount of liquid alcohol on an absorbent material either at the top or at the bottom of a vial and then allowing the alcohol to vaporize with time 41,42 . As the concentration of alcohol vapor increases with time, behavioral responses can be assessed.…”

The Genetics of Alcohol Responses of Invertebrate Model Systems

“…In this case, loss of climbing ability or effects on total movement can be assessed by monitoring the flies' positions from a series of time-lapse photographs (Pohl et al, 2013). Akinesia, or lack of motion, has sometimes been used as a proxy for loss of postural control and sedation (Park, Sedore, Cronmiller, & Hirsh, 2000;Wen et al, 2005).…”

“…Animals are said to have acquired tolerance if preexposed animals respond less than naïve animals. Ethanol tolerance has been quantified by monitoring sedation, recovery from sedation, elution from the in ebriometer, ethanol-induced hyperactivity, and learning (Berger et al, 2004;Bhandari et al, 2009;Kong, Allouche, et al, 2010;Kong, Woo, et al, 2010;Pohl et al, 2013;Scholz et al, 2000). Commonly measured mammalian phenotypes such as hypothermic and anxiolytic effects of ethanol are not used in invertebrates.…”

“…A mutation in any one of these genes causes flies to be arrhythmic. Pohl et al (2013) showed that mutations in these genes did not alter ethanol sensitivity. However, a mutation in per or tim or cyc eliminated the capacity to acquire functional ethanol tolerance, but a mutation in Clk did not.…”

The Epigenetic Landscape of Alcoholism

“…The class III HDACs, sirtuins, due to their NAD + dependency, are believed to be critical in the regulation of biological and circadian rhythms, cell survival, metabolic activity, aging and neurodegeneration (Hirayama et al, 2007; Nakahata et al, 2008; Bishop & Guarente, 2007; Cohen et al, 2004; Kim et al, 2007). Dysregulation of the circadian clock has been implicated in psychiatric disorders and alcohol phenotypes in both vertebrate and invertebrate systems (Mansour et al, 2006; Pohl et al, 2013; Sarkar, 2012). Hence HDAC manipulation thereby affecting circadian pathways, could be an important avenue for therapeutic intervention.…”