Circadian genes differentially affect tolerance to ethanol in Drosophila

Abstract

Background: There is a strong relationship between circadian rhythms and ethanol (EtOH) responses. EtOH consumption has been shown to disrupt physiological and behavioral circadian rhythms in mammals (Alcohol Clin Exp Res 2005b, 29, 1550). 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 toward EtOH tolerance and whether rhythmicity itself is necessary for tolerance.

Methods: We tested flies carrying mutations in core clock genes for the capacity to acquire EtOH 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 EtOH vapor to determine if other facets of the EtOH response were affected by the mutations. Gas chromatography was used to measure internal EtOH concentration. Constant light was used to nongenetically 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 EtOH absorption.

Conclusions: Mutations affecting different parts of the intracellular circadian clock can block the capacity to acquire rapid EtOH tolerance. However, the role of circadian genes in EtOH tolerance is independent of their role in producing circadian rhythmicity. The interference in the capacity to acquire EtOH 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 EtOH tolerance.

Keywords: Alcohol Tolerance; Circadian Rhythm; Drosophila; Mutation.

Figure 1. Circadian mutants do not affect sensitivity to ethanol

Knockdown curves are used to compare the ethanol sensitivity of the wild type and the various backcrossed circadian mutant alleles. In each plot, the sensitivity of the wild-type Canton S line is compared with a circadian mutant that has been backcrossed into the wild-type background. Shown are A) per⁰¹, C) tim⁰¹, E) cyc⁰¹, and G) Clk^JRK. Bar graphs compare the time required for 50% to be knocked down (K₅₀) by the ethanol vapor from a 35% ethanol solution for B) per⁰¹, D) tim⁰¹, F) cyc⁰¹, and H) Clk^JRK. None of the comparisons were significantly different. Statistical significance was determined using Student's t test with a cutoff of p<0.05. n=4 vials for per⁰¹ test, n=6 vials for all others. Each vial contains 10 animals. I) The K₅₀ occurs at a specific internal ethanol concentration. Thirty-nine ethanol sedations (10 flies per sedation) were performed on different days or with different ethanol concentrations (35% or 40% ethanol was used). This produced wide variation in the K50 value (29 minutes to 71 minutes). When half of the flies were sedated (K₅₀) the flies were sacrificed and the internal ethanol concentration measured (111 mM +/−2.3, N=39). The slope of the best fit line does not differ significantly from zero (r²<0.002, P>0.8). J) The CS (wild type) and mutant per⁰¹, tim⁰¹, cyc⁰¹, and Clk^JRK stocks do not differ from one another in their internal ethanol hemolymph concentration after vapor treatment. Internal ethanol content was determined by gas chromatography after 30 minutes of exposure to the vapor from a 35% ethanol solution. Ethanol treatment is identical to panes A–H except that all stocks were treated at the same time. The lack of statistical significance was determined using one way Anova with the Dunnett's Multiple Comparison Test. Error bars are standard error of the mean, n= 6 vials. Each vial contains 10 animals.

Figure 2. Some circadian mutants disrupt tolerance to ethanol

Shown are knockdown curves that compare flies receiving their first sedation to flies receiving their second sedation for A) CS, B) per⁰¹, C) tim⁰¹, D) cyc⁰¹, and E) Clk^JRK. The corresponding bar graph for K₅₀ is shown for F) CS, G) per⁰¹, H) tim⁰¹, I) cyc⁰¹, and J) Clk^JRK. Statistical significance was determined using Student's t test (* p<0.05, **p<0.01; n=4 vials for per⁰¹ test, n=6 vials for all others; each vial contains 10 animals).

Figure 3. A movement assay shows distinct attributes of tolerance

A) An activity plot of wild-type Canton S flies that compares the number of flies moving after receiving their first or second dose of ethanol. B) A baseline movement activity plot of the wild type flies placed in the test chamber without ethanol vapor. The baseline flies were mock-sedated 24 h before being placed in the test chamber. The baseline after ETOH flies were sedated with ethanol 24 h before being placed in a test chamber without ethanol. C) The ethanol component of the response curve was isolated by subtracting the baseline data (panel B) from the raw ethanol response data (panel A). The 1st minus baseline curve is produced by subtracting the baseline curve (panel B) from the 1st sedation curve (panel A). The 2nd minus baseline after ethanol curve was produced by subtracting the baseline after ETOH (panel B) from the 2nd sedation data (panel A). Error bars are standard error of the mean (n=6). The dotted white line is a nonlinear best-fit of single Gaussian curve to the data. Bar graphs were derived from the data in panel C and depict D) the T₅₀ of the rising phase, which is the time at which the rises to 50% maximum amplitude, E) the time of maximal movement activity (T_max), and F) the T₅₀ of the falling phase, which is the time at which the curve decays to 50% maximum amplitude. The wild type shows evidence that it has acquired ethanol tolerance in all three of these parameters. Statistical significance was determined using a two-tailed Student's t test (**p=0.0016; ***p<0.001; n=6 vials; each vial contains 10 animals).

Figure 4. A movement assay shows that flies carrying the per⁰¹ mutation do not acquire any attributes of rapid tolerance

A) An activity plot that compares the number of flies moving for per⁰¹ flies that are receiving their first or second dose of ethanol. B) A baseline movement activity plot of per⁰¹ flies was generated as described in figure 2. C) The ethanol component of the response curve was isolated as described in figure 2. The per⁰¹ mutants do not show evidence of rapid ethanol tolerance in the the T₅₀ of the rising phase of the activity curve (D), the T_max of the activity curve (E), or in the T₅₀ of the falling phase of the activity curve (F). Statistical significance was determined using a two-tailed Student's t test (n=4 vials; each vial contains 10 animals).

Figure 5. A movement assay shows that flies carrying the tim⁰¹ mutation do not acquire any attributes of rapid tolerance

A) An activity plot that compares the number of flies moving for tim⁰¹ flies receiving their first or second dose of ethanol. B) A baseline movement activity plot of tim⁰¹ flies was generated as described in figure 2. C) The ethanol component of the response curve was isolated as described in figure 2. The tim⁰¹ mutants do not show evidence of rapid ethanol tolerance in the the T₅₀ of the rising phase of the activity curve (D), the T_max of the activity curve (E), or in the T₅₀ of the falling phase of the activity curve (F). Statistical significance was determined using a two-tailed Student's t test (n=6 vials; each vial contains 10 animals).

Figure 6. A movement assay shows that the cyc⁰¹ mutation disrupts some but not all aspects of the tolerance response

A) An activity plot that compares the number of flies moving for cyc⁰¹ flies receiving their first or second dose of ethanol. B) A baseline movement activity plot of cyc⁰¹ flies was generated as described in figure 2. C) The ethanol component of the response curve was isolated as described in figure 2. The cyc⁰¹ mutants do not show evidence of rapid ethanol tolerance in the the T₅₀ of the rising phase of the activity curve (D), the T_max of the activity curve (E), or in the T₅₀ of the falling phase of the activity curve (F). Statistical significance was determined using a two-tailed Student's t test (n=6 vials; each vial contains 10 animals).

Figure 7. A movement assay shows that the Clk^JRK mutation does not disrupt tolerance

A) An activity plot that compares the number of flies moving for Clk^JRK flies receiving their first or second dose of ethanol. B) A baseline movement activity plot of Clk^JRK flies was generated as described in figure 2. C) The ethanol component of the response curve was isolated as described in figure 2. The Clk^JRK mutants show evidence that they have acquired rapid ethanol tolerance in the T₅₀ of the rising phase of the activity curve (D), the T_max of the activity curve (E), or in the T₅₀ of the falling phase of the activity curve (F). Statistical significance was determined using a two-tailed Student's t test (**p<0.005; n=6 vials; each vial contains 10 animals).

Figure 8. Constant light eliminates ethanol tolerance in wild-type flies

Knockdown curves are shown comparing flies receiving their first sedation to flies receiving their second sedation for A) flies maintained in a 12:12 light:dark cycle and C) flies maintained in constant light. Corresponding bar graphs depicting K₅₀ comparing flies receiving their first and second treatment are shown for B) Light:Dark flies and D) Constant light flies. Under 12:12 light dark conditions the rhythm index measured over three days was 0.139+/− 0.011 (n=24) while under constant light conditions the rhythm index dropped to 0.041 +/− 0.0009 (n=23). Statistical significance was determined using Student's t test (**p<0.01; n=6 vials; each vial contains 10 animals).