A Low Concentration of Ethanol Impairs Learning but Not Motor and Sensory Behavior in Drosophila Larvae

Drosophila melanogaster has proven to be a useful model system for the genetic analysis of ethanol-associated behaviors. However, past studies have focused on the response of the adult fly to large, and often sedating, doses of ethanol. The pharmacological effects of low and moderate quantities of ethanol have remained understudied. In this study, we tested the acute effects of low doses of ethanol (∼7 mM internal concentration) on Drosophila larvae. While ethanol did not affect locomotion or the response to an odorant, we observed that ethanol impaired associative olfactory learning when the heat shock unconditioned stimulus (US) intensity was low but not when the heat shock US intensity was high. We determined that the reduction in learning at low US intensity was not a result of ethanol anesthesia since ethanol-treated larvae responded to the heat shock in the same manner as untreated animals. Instead, low doses of ethanol likely impair the neuronal plasticity that underlies olfactory associative learning. This impairment in learning was reversible indicating that exposure to low doses of ethanol does not leave any long lasting behavioral or physiological effects

A DNA Element Regulates Drug Tolerance and Withdrawal in Drosophila

Drug tolerance and withdrawal are insidious responses to drugs of abuse; the first increases drug consumption while the second punishes abstention. Drosophila generate functional tolerance to benzyl alcohol sedation by increasing neural expression of the slo BK-type Ca2+ activated K+ channel gene. After drug clearance this change produces a withdrawal phenotype—increased seizure susceptibility. The drug-induced histone modification profile identified the 6b element (60 nt) as a drug responsive element. Genomic deletion of 6b produces the allele, sloΔ6b, that reacts more strongly to the drug with increased induction, a massive increase in the duration of tolerance, and an increase in the withdrawal phenotype yet does not alter other slo-dependent behaviors. The 6b element is a homeostatic regulator of BK channel gene expression and is the first cis-acting DNA element shown to specifically affect the duration of a drug action.This work has been supported by National Institutes of Health (NIH) R01 AA018037 (subgroup: NIAAA, title: Epigenetic Modification as a Mechanism to Produce Functional Tolerance). URL: http://www.niaaa.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Waggoner Center for Alcohol and Addiction ResearchEmail: [email protected]

Modification of the <i>slo</i> transcriptional control region.

<p><b>A</b>) Transcriptional control region of <i>slo</i>. Labelled arrows are tissue-specific transcription start sites of the previously-described promoters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B11" target="_blank">11</a>]. Blue boxes represent alternative 5' exons that are unique products of each promoter. Rightmost box on the line represents the first exon common to all <i>slo</i> transcripts. Neuronal splice variants begin translation in this exon. The remainder of the coding region is not shown. Boxes below the line are non-coding conserved DNA elements [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B11" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B40" target="_blank">40</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B41" target="_blank">41</a>]. The table summarizes benzyl alcohol induced histone H4 hyper-acetylation, neural expression of <i>slo</i>, and functional tolerance (data from Wang et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B6" target="_blank">6</a>]). Plusses correspond to the H4 hyperacetylation. Arrows reflect relative abundance of <i>slo</i> mRNA (RNA). Checks identify when behavioral tolerance can be detected in the first 48 h (Tol). <b>B</b>) Homologous recombination occurred between the replacement DNA (red) and its chromosomal counterpart (blue) replaces 6b with a floxed mini-white gene. Cre recombinase was used to excise the loxP-flanked <i>white</i> gene to produce the <i>slo</i>^∆6b allele in which the 6b element has been replaced by a loxP site. <b>C</b>) Crossing scheme for 6b targeting. <b>D</b>) Products produced at the different steps in the crossing scheme described in panel C<b>. E</b>) Southern blotting confirms homologous recombination into the <i>slo</i> locus. Restriction maps of wild type, <i>slo</i>^w∆6b<i>, and </i><i>slo</i>^∆6b. Probe indicated above maps. The recipient line (WT), produces an 8.5 kb band. The <i>slo</i>^w∆6b and <i>slo</i>^∆6b recombinants produce bands whose size is indicative of high fidelity homologous recombination into at the position of the 6b element (cf. panels D and E).</p

The <i>slo</i>^∆6b mutant shows unusually long-lived benzyl alcohol tolerance.

<p><b>A</b>) Schematic of the paradigm used to determine the time course of functional tolerance. A stock shows tolerance if it recovers more rapidly from its second sedation than from its first sedation. A population of age-matched females were separated into two groups. One group was mock sedated and the second group was sedated with benzyl alcohol. The animals were then housed in separate vials with food for the time intervals in panel B (1d -28 d) and then both groups were benzyl alcohol sedated in tandem, moved to fresh air (t=0), and their recovery recorded. <b>B</b>) The recovery curves describe the percentage of flies recovering after a single sedation (blue) and after a two sequential sedations (red) that were separated by the time interval shown. Tolerance lasts for at least 28 d in <i>slo</i>^∆6b; however, it is detected in wild type for only a week. Error bars represent SEM, but significance difference between curves is determined by log-rank analysis (n=4–6. *<i>P</i> ≤ 0.05).</p

The <i>slo</i>^∆6b mutation specifically affects drug-induced neural expression of <i>slo</i>.

<p><b>A</b>) Six hours after benzyl alcohol sedation induction slo expression is increased in the wild type (CS) and <i>slo</i>^∆6b mutants; however, induction is greater in the mutant than in the wild type. <b>B</b>) The relative abundance of <i>slo</i> mRNA was not statistically different from the baseline abundance 24 h after sedation for either the wild type or the <i>slo</i>^∆6b mutant. Unpaired <i>t</i>-test, n=3. Error bars represent SEM.</p

Electrophysiological analysis of wild type and <i>slo</i>^∆6b stocks.

<p><b>A</b>) Electroconvulsive stimuli (ES) induce a stereotypical seizure response from the giant fiber pathway. Constant low-frequency stimulations were applied continuously to assess the state of responsivity of the giant fiber pathway. A seizure consists of a high-frequency initial discharge (ID) followed by a period in which the giant fiber fails to respond to stimulation (Failure) followed by a delayed discharge (DD) and then by a recovery of normal responsivity. The seizure threshold was identified by delivering electroconvulsive shock of varying voltage until the production of a stereotypical seizure response. <b>B</b>) The <i>slo</i>^∆6b mutant exhibited a significant lower average seizure threshold compared to wild-type flies. Benzyl alcohol sedation reduced stimulus voltage in both stocks. <b>C</b>) The drug-induced reduction in seizure threshold was greater in <i>slo</i>^∆6b mutants than in wild-type animals one day after sedation. <b>D</b>,<b>E</b>) The average seizure stimulus voltages of both WT and <i>slo</i>^∆6b returned to baseline at 7 d after sedation. <b>F</b>) Time course of seizure threshold after drug sedation. Re-plot of the data presented in panels B-E to illustrate the convergence of seizure threshold as a function of age. Wild-type (WT) and <i>slo</i>^∆6b lines are compared before and after benzyl alcohol (BA) sedation. Unpaired Student’s t-test, P values and number of repeats as shown. Error bars show SEM.</p

State of histone H4 acetylation across the <i>slo</i> transcriptional control region after benzyl alcohol sedation.

<p><b>A</b>) Map of the <i>slo</i> transcriptional control region and areas assayed by the chromatin immunoprecipitation assay. Arrowheads identify the position of the tissue-specific <i>slo</i> core promoters, and open boxes on the line represent exons. The gray boxes below the line show the conserved elements tested in the chromatin immunoprecipitation assay. In <i>slo</i>^∆6b, the 6b site was replaced by a loxP element. <b>B</b>) H4 acetylation levels 6 h detected in WT and <i>slo</i>^∆6b after benzyl alcohol sedation. One-way ANOVA with Dunnett’s comparison post test. n=3. From left to right * signifies P=0.0458, 0.0278, and 0.0200. *** signifies P=0.0008. Fold change of acetylation was the ratio of the acetylation levels of drug-sedated flies over untreated ones. <b>C</b>) Acetylation state surveyed 24 h after BA sedation. One-way ANOVA with Dunnett’s comparison post test. n=3. From left to right * signifies P=0.0387, 0.0486, and 0.0239. Error bars represent SEM.</p

The time course analysis of <i>slo</i> transcription in <i>slo</i>^∆6b flies.

<p>The mRNA levels of <i>slo</i> were determined by real-time RT-PCR using C1 primers that amplify only neural <i>slo</i> transcripts. One-way ANOVA with Dunnett’s comparison post test, n=3. Error bars represent SEM.</p

<i>slo</i>^∆6b flies are normal with respect to most behaviors.

<p>The <i>slo</i>^∆6b strain was backcrossed to the Canton S wild-type stock (WT) for six generations prior to analysis. The locomotor activity, (<b>A</b>), walking speed (<b>B</b>), climbing (<b>C</b>) and flight (<b>D</b>) of <i>slo</i>^∆6b are similar to WT, while the <i>slo</i>⁴ null mutants are deficient in all four behaviors. <b>E</b>) Circadian activity. LD entrained flies were transferred to DD and monitored. WT and <i>slo</i>^∆6b flies demonstrated rhythmic oscillation in activity. <i>slo</i>^∆6b had slightly higher peak activity than WT. However, the <i>slo</i>⁴ null mutants were arrhythmic. n=25. <b>F</b>) Percentage of WT, <i>slo</i>^∆6b, and <i>slo</i>⁴ identified as rhythmic. <b>G</b>) <i>slo</i>^∆6b mutants had a normal circadian period length. n=25. <b>H</b>) The <i>slo</i>^∆6b mutation does not alter drug resistance. Age-matched females were BA sedated and their recovery rate was determined. n=6. <b>I</b>) The <i>slo</i>^∆6b mutation, does not disturb the splicing out of the intron in which it is located. RT-PCR shows that the flanking exons are spliced normally. <b>J</b>) The <i>slo</i>^∆6b allele shows normal basal expression level (P=0.859; n=3). mRNA abundance was measured by RT-qPCR using primers specific for neuronal <i>slo</i> transcripts. <b>Statistical tests</b>. A, B, C, and F–one-way ANOVA with Dunnett’s comparison post test. *** indicates P≤ 0.001. N=25 (A), 3 (B), and 4 (C). For H, the log-rank test was used to evaluate significance between the recovery curves. For J, an unpaired <i>t</i>-test was used to evaluate significance. All error bars are SEM.</p