Dose-dependent alterations in ethanol-induced hyperactivity and sedation sensitivity in a rapid ethanol tolerance paradigm. (A) Exposure scheme for induction and detection of ethanol rapid tolerance. Groups of genetically identical flies are given two 26-minute exposures to ethanol vapor, separated by a 3.5-hour rest under standard culturing conditions. (B) Prior to ethanol exposure (“A” on horizontal axis) flies exhibit low levels of locomotor activity. Exposure of naive flies to a continuous stream of ethanol vapor (47% concentration, open box) results in an olfactory startle response that subsides by 1 minute, followed by a hyperactive phase from approximately 2 to 25 minutes (Exp 1). Hyperactivity declines with increasing locomotor incoordination and sedation. Following a 3.5-hour rest, flies exposed a second time to the same ethanol vapor concentration exhibit a small increase in hyperactivity during its onset (Exp 2). (C and D) Exposure to higher concentration ethanol vapor results in greater differences in the hyperactive phase between Exp 1 and Exp 2. (E) Distance traveled (Dist) from 2 to 25 minutes ethanol vapor exposure. Distance traveled was significantly greater during Exp 2 versus Exp 1 (47%: *p = 0.037, 60%: **p < 0.0005, 73%: **p < 0.0005, paired t-test, n = 15). (F) Change in distance traveled (ΔDist) between Exp 2 and Exp 1 (**p < 0.0005, ANOVA, all samples different by Tukey’s multiple comparison test). (G) Time course of ethanol sedation at 60% ethanol vapor. (H) The fraction of flies sedated (Sed) following 26-minute ethanol exposure. Fewer flies were sedated following the second ethanol exposure at all concentrations tested (47%: *p = 0.038, 60%: **p < 0.0005, 73%: **p < 0.0005, paired t-test, n = 15). (I) Sedation tolerance (Sed Tol) is the difference in the fraction of flies sedated between Exp 1 and Exp 2 (**p < 0.0005, ANOVA, 47% sample different by Tukey’s multiple comparison test).

Time course microarray analysis of gene expression levels following a single exposure to a moderate dose of ethanol. (A) Treatment scheme for microarray analysis. Fourteen groups of 200 flies were exposed to either 60% ethanol vapor or humidified air for 30 minutes, allowed to recover for 0 to 210 minutes and then frozen at the time-point indicated by the triangles. Two additional groups were frozen as no exposure controls (time 0). RNA extracted from the heads of the resulting 16 samples was hybridized to Drosophila 2.0 microarrays (Affymetrix). (B) Clustered gene expression patterns (rows) for exposure to ethanol vapor (left) and humidified air (right) at the given time-points (columns), represented as the medoid values for each cluster. Green and magenta indicate reduced and increased expression relative to the averaged unexposed controls, respectively. (C to F) Example expression level time courses from the microarray analysis (C and E) and qPCR confirmations using RNA isolated from a separate exposure series (D and F) for the genes tyramine decarboxylase 1 (Tdc1; C and D) and Sir2 (E and F). Data are expressed as fold change relative to unexposed controls. Time of the 30-minute ethanol or air exposure is indicated as a box on the horizontal axis. E (red) and A (blue) indicate gene expression levels in ethanol- and air-exposed samples, respectively. Error bars indicate standard deviation of 3 sample replicates for qPCR. (G) All genes (rows) in cluster 1. Genes in area bracketed by red bars are predominantly olfactory specific, showing a large, coordinate decrease in expression of many, but not all, olfactory specific genes. (H) Overlap of genes identified in this and 2 previous microarray studies (Morozova et al., 2006 and Urizar et al., 2007) of gene regulation by ethanol exposure in Drosophila.

Correlation of behavioral measures for ethanol tolerance in strains surveyed for ethanol behavioral alterations. Graph depicts values for all strains surveyed (gray dots) for ΔDist on the horizontal axis, and Sed Tol on the vertical axis. Linear regression analysis (line) indicated a moderate correlation between ΔDist and Sed Tol.

Behavioral effects of mutation of AcCoAS. (A) Expression levels by microarray analysis of AcCoAS following exposure to ethanol (squares) or air (diamonds) relative to untreated controls (time 0). (B) The AcCoAS locus consists of 3 transcriptional units (RA, RB, RC; shaded region indicates extent of open reading frame). The transposon f03474 is inserted just upstream of the RB transcription start site. The probeset used to detect AcCoAS transcripts by qPCR spanned the last intron. (C) Locomotor activity profile for AcCoAS^f03474 compared with matched controls (Berlin) during two 26-minute exposures to 60% ethanol vapor separated by a 3.5-hour rest. Locomotor activity in humidified air (A) is measured just prior to ethanol exposure (0 to 26 minutes); n = 6. (D to G) Ethanol behavioral measures derived for experimental data depicted in C (D: *p = 0.014; E: p = 0.152; F: p = 0.134; G: p = 0.371, paired t-test).

Behavioral effects of deletion of Or83b. (A) Locomotor activity profile of Or83b¹ compared with matched Berlin controls. n = 6. Inset: expression levels by microarray analysis of Or83b following exposure to ethanol or humidified air relative to untreated controls (time 0). (B to E) Measures of ethanol responses derived from experimental data depicted in A (B: p = 0.051; C: *p = 0.032; D: p = 0.085; E: p = 0.581, paired t-test).

Behavioral effects of disruption of Spn27A. (A) Expression levels by microarray analysis of Spn27A following exposure to ethanol or humidified air relative to untreated controls (time 0). (B) The Spn27A locus consists of a single transcriptional unit with no introns and a single open reading frame (shaded). The transposon e02539 is inserted into the open reading frame. (C) Locomotor activity profile for Spn27A^e02539, compared with matched Berlin controls; n = 6. (D to G) Measures of ethanol responses derived from the experimental data depicted in C (D: **p = 0.001; E: p = 0.994; F: p = 0.092; G: p = 0.431; paired t-test).

Behavioral effects of deletion of Sir2. (A) The Sir2 locus consists of a single transcriptional unit that is predicted to overlap with the DnaJ-H gene at the 5′-end of both genes. The 2A–7–11 targeted deletion, depicted below the Sir2 locus, removes the Sir2 translation start site and the majority of the open reading frame (shaded). Probes for qPCR are indicated as bars above the genes. The NP1145 transposon insertion is located just upstream of the Sir2 translation start site. (B) Transcript levels of DnaJ-H are unaffected in 2A–7–11 homozygotes, when compared with genetic background controls. (C) Loco-motor activity profile for Sir2^2A–7–11, compared with matched Berlin controls, tested in the rapid tolerance paradigm and exposed to 60% ethanol vapor; n = 8. (D to G) Measures of ethanol responses derived from the experimental data depicted in C (D: p = 0.562; E: *p = 0.027; F: **p = 0.006; G: *p = 0.027; 2-sample t-test with pooled variance). (H) Locomotor activity profile for Sir2^2A–7–11, compared with matched Berlin controls, tested in the rapid tolerance paradigm and exposed to 73% ethanol vapor; n = 3. (I to L) Measures of ethanol responses derived from the experimental data depicted in H (I: p = 0.729; J: **p = 0.003; K: p = 0.225; L: p = 0.697; paired t-test).