Ethanol sensitivity and tolerance in long-term memory mutants of Drosophila melanogaster

Abstract

Background: It has become increasingly clear that molecular and neural mechanisms underlying learning and memory and drug addiction are largely shared. To confirm and extend these findings, we analyzed ethanol-responsive behaviors of a collection of Drosophila long-term memory mutants.

Methods: For each mutant, sensitivity to the acute uncoordinating effects of ethanol was quantified using the inebriometer. Additionally, 2 distinct forms of ethanol tolerance were measured: rapid tolerance, which develops in response to a single brief exposure to a high concentration of ethanol vapor; and chronic tolerance, which develops following a sustained low-level exposure.

Results: Several mutants were identified with altered sensitivity, rapid or chronic tolerance, while a number of mutants exhibited multiple defects.

Conclusions: The corresponding genes in these mutants represent areas of potential overlap between learning and memory and behavioral responses to alcohol. These genes also define components shared between different ethanol behavioral responses.

Fig. 1

Ethanol sensitivity of the Drosophila memory mutants. (A) Histogram illustrates frequency distribution of inebriometer mean elution times (MET, min; 3-minute bins) for 52 memory mutants. Solid line indicates mutant population mean MET, 19.5 minutes, and dashed line the control strain MET, 20.9 ± 0.2 minutes. (B) Bar graph represents inebriometer METs (±SEM) of the 8 mutants showing altered ethanol sensitivity in first-round analysis (Ctl, control; *denotes p < 0.001 for each mutant vs. Ctl; F_52,198 = 10.45, p < 0.001). (C and D) Individual inebriometer elution profiles for the most ethanol-sensitive mutant, ikar, versus control (C) and for the ethanol-resistant mutant, murashka-2, versus control (D); n = 4 (here and elsewhere, n represents the number of samples, not the number of flies; in all figures, error bars represent standard errors of the mean).

Fig. 2

Additional ethanol sensitivity mutants identified by retesting. Inebriometer assays were performed on small groups of 1 to 3 candidate mutants plus control (Ctl); p values for each mutant versus Ctl are denoted by asterisks: *p < 0.05; **p < 0.001; n = 4. (A) Only barbos-1 was significantly different from control (F_3,15 = 4.99, p = 0.018; barbos-1 vs. Ctl, p = 0.013). (B) Neither novichok nor valiet-3 was significantly different from control (F_2,11 = 5.22, p = 0.03; novichok vs. Ctl, p = 0.24; valiet-3 vs. Ctl, p = 0.08). (C) Both joy and milord-1 showed increased ethanol sensitivity (F_2,11 = 32.17, p < 0.001), as did milord-2 (D) (F_1,7 = 47.7, p < 0.001), while krasavietz (E) exhibited significantly reduced sensitivity (F_1,7 = 48.05, p < 0.001).

Fig. 3

Rapid ethanol tolerance in the memory mutants. Flies were pre-exposed for 30 minutes to ethanol vapor (at a relative flow rate of 60/40 E/A, a sedating dose) or to humidified air at an equivalent total flow rate as a control, allowed to recover for 3.5 hours and assayed in the inebriometer. (A) Histogram represents frequency distribution of values for rapid tolerance (minutes, min.), using 3 minute bins. Vertical solid line denotes mutant population mean tolerance (8.8 minutes), and dashed line rapid tolerance of the control strain (Ctl, 10.2 ± 0.9 minutes). (B) MET and rapid tolerance values for krasavietz, milord-1, and valiet-3, the 3 reduced rapid tolerance mutants identified in first-round analysis (F_52,198 = 5.83, p < 0.001; *denotes p < 0.001 for each mutant vs. Ctl; n = 4). For MET values, dark gray bars denote flies pre-exposed to ethanol vapor, and light gray bars, humidified air (treatment control). (C and D) Average inebriometer elution profiles for Ctl flies (C) and the krasavietz mutant (D). “Ethanol” denotes flies pre-exposed to ethanol vapor, and “Air” the treatment control, air pre-exposed flies.

Fig. 4

Additional rapid tolerance mutants identified by retesting. Inebriometer assays measuring tolerance were performed on small groups of 1 to 3 candidate mutants plus control (Ctl). Dark gray bars denote MET values for flies pre-exposed to ethanol vapor, and light gray bars, humidified air; p values for tolerance of each mutant compared with Ctl are denoted by asterisks: *p < 0.01; **p < 0.001; n = 4–6. The mutants valiet-1 (A) (F_1,7 = 41.38, p < 0.001) and rogdi (B) (F_3,17 = 5.53, p = 0.01; rogdi vs. Ctl, p = 0.0013) showed reduced tolerance, while milord-2 was not significantly different from Ctl (C) (F_1,7 = 0.9; p = 0.38). (D) The mutants ikar, ruslan and zolotistuy also exhibited significant reductions in rapid tolerance (F_4,25 = 6.96, p < 0.001).

Fig. 5

Chronic ethanol tolerance in the memory mutants. Flies were pre-exposed overnight (20 to 28 hours) to a nonsedating concentration of ethanol vapor (10/80 E/A) or to humidified air as a control and assayed in the inebriometer. (A) Histogram represents frequency distribution of values for chronic tolerance (minutes, min.), using 2-minute bins. Vertical solid line denotes mutant population mean chronic tolerance (5.6 minutes), and dashed line denotes tolerance of the control strain (Ctl, 6.8 ± 1.3 minutes). (B) MET and chronic tolerance values for the control strain (Ctl) and the mutant with the lowest chronic tolerance, jack. Dark gray bars denote MET values for flies pre-exposed to ethanol, and light gray bars, humidified air. In first-round analysis across the entire collection, no mutants exhibited chronic tolerance significantly different from Ctl (F_52,272 = 4.27; p < 0.001; n = 4–10). (C and D) Average inebriometer elution profiles for Ctl (C) or jack (D) flies pre-exposed overnight to low-level ethanol vapor (Ethanol) or to an equivalent flow rate of humidified air (Air).

Fig. 6

Chronic tolerance mutants identified by small-group retesting. Inebriometer assays measuring tolerance were performed on small groups of 1 to 3 candidate mutants plus control (Ctl). (A–D) Dark gray bars denote MET values for flies pre-exposed to ethanol, and light gray bars, humidified air; p values for tolerance of each mutant versus Ctl are denoted by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001; n = 5–9. (A) The mutant john exhibited significantly increased chronic tolerance (F_2,21 = 10.0, p < 0.001), while (B–D) a total of 8 mutants exhibited significantly reduced tolerance [(B) F_3,26 = 6.77, p = 0.002; (C) F_2,15 = 6.45, p = 0.011; (D) F_3,22 = 7.42, p = 0.002)].

Fig. 7

Ethanol pharmacokinetics of ethanol sensitivity and tolerance mutants. (A and B) Mutants exhibiting altered ethanol sensitivity were exposed to ethanol vapor at a relative flow rate of 60/40 (A) or 55/45 E/A (B) for 30 minutes and processed immediately. No significant difference in ethanol content was seen for any of the altered sensitivity mutants [(A), F_7,31 = 1.02, p = 0.44; (B) F_6,29 = 2.38, p = 0.06; n = 4–6)]. (C) Mutants exhibiting reduced rapid tolerance were pretreated by a 30-minute exposure to 60/40 E/A (Ethanol), or to humidified air at an equivalent flow rate (Air). Following a 3.5-hour recovery, all samples were exposed for 30 minutes to 55/45 E/A and processed. In two-way ANOVA, a significant main effect of genotype was seen (F_8,79 = 6.78, p < 0.001), with the mutant milord-1 exhibiting a significant decrease in ethanol content compared with Ctl (p < 0.001); no significant effect of pre-exposure treatment was observed (F_1,79 = 0.50, p = 0.48), and there was no significant interaction between genotype and pre-exposure treatment (F_8,79 = 0.38, p = 0.93). n = 4–8. (D) Mutants exhibiting altered chronic tolerance were pretreated by ∼24-hour exposure to 10/80 E/A (Ethanol) or to humidified air at an equivalent flow rate (Air), then all samples were exposed for 30 minutes to 55/45 E/A and processed. In two-way ANOVA, a significant effect of genotype was detected (F_9,89 = 12.03, p < 0.001), with the mutants rafael and ruslan significantly different from Ctl (*p < 0.001). A significant effect of pre-exposure condition was also detected (F_1,89 = 26.2, p < 0.001), and there was a significant interaction between genotype and pre-exposure condition (F_9,89 = 2.56, p < 0.013) for the mutants jack (p < 0.001), john (p = 0.017) and ruslan (p < 0.001; significance of interaction is denoted by daggers: ^†p < 0.05; ^††p < 0.001; n = 3–8). (E) Line diagram illustrates exposure regimens for sensitivity and tolerance mutants; arrow indicates point at which flies were processed to determine ethanol content.