Rapid and Chronic Ethanol Tolerance Are Composed of Distinct Memory-Like States in Drosophila

Abstract

Ethanol tolerance is the first type of behavioral plasticity and neural plasticity that is induced by ethanol intake, and yet its molecular and circuit bases remain largely unexplored. Here, we characterize the following three distinct forms of ethanol tolerance in male Drosophila: rapid, chronic, and repeated. Rapid tolerance is composed of two short-lived memory-like states, one that is labile and one that is consolidated. Chronic tolerance, induced by continuous exposure, lasts for 2 d, induces ethanol preference, and hinders the development of rapid tolerance through the activity of histone deacetylases (HDACs). Unlike rapid tolerance, chronic tolerance is independent of the immediate early gene Hr38/Nr4a Chronic tolerance is suppressed by the sirtuin HDAC Sirt1, whereas rapid tolerance is enhanced by Sirt1 Moreover, rapid and chronic tolerance map to anatomically distinct regions of the mushroom body learning and memory centers. Chronic tolerance, like long-term memory, is dependent on new protein synthesis and it induces the kayak/c-fos immediate early gene, but it depends on CREB signaling outside the mushroom bodies, and it does not require the Radish GTPase. Thus, chronic ethanol exposure creates an ethanol-specific memory-like state that is molecularly and anatomically different from other forms of ethanol tolerance.SIGNIFICANCE STATEMENT The pattern and concentration of initial ethanol exposure causes operationally distinct types of ethanol tolerance to form. We identify separate molecular and neural circuit mechanisms for two forms of ethanol tolerance, rapid and chronic. We also discover that chronic tolerance forms an ethanol-specific long-term memory-like state that localizes to learning and memory circuits, but it is different from appetitive and aversive long-term memories. By contrast, rapid tolerance is composed of labile and consolidated short-term memory-like states. The multiple forms of ethanol memory-like states are genetically tractable for understanding how initial forms of ethanol-induced neural plasticity form a substrate for the longer-term brain changes associated with alcohol use disorder.

Keywords: Drosophila; alcohol; ethanol; genetics; memory; tolerance.

Figure 1.

Types of ethanol tolerance in Drosophila. A, Ethanol exposure schemes to induce and measure ethanol sensitivity and tolerance. Challenge doses (C) are 55% ethanol unless noted otherwise. Gene expression measurements were sampled 1 h after the ethanol challenge dose. B, Dose response for chronic ethanol pre-exposure. Chronic tolerance is ST50, chronic minus acute. C, Ethanol absorption and metabolism with air or 16% chronic ethanol pre-exposure. Flies were exposed to 30 min of 20% ethanol to avoid sedation. Absorption was measured immediately afterward, and metabolism was measured 30 min later. D, Left, Chronic ethanol pre-exposure caused resistance to sedation. Right, Chronic ethanol exposure induces tolerance that lasts for 2 d. E, Chronic ethanol exposure interfered with the subsequent development of rapid tolerance. Flies were either given 48 h of chronic ethanol exposure or humidified air, and 24 h later were subjected to a rapid tolerance test, two 30 min 60% EtOH exposures, E1 and E2, 4 h apart. Rapid tolerance is ST50, E2–E1. F, Chronic ethanol exposure induced ethanol preference, measured in the CAFÉ two-choice assay. G, Left, Repeated inebriating doses of ethanol caused resistance to ethanol sedation. Right, Repeated ethanol exposure induced tolerance that lasts for at least 1 d. Tolerance is from data shown in the left panel, repeated minus acute. H, Repeated ethanol exposure inhibited the subsequent development of rapid tolerance. I, Repeated ethanol exposure induced ethanol aversion in the CAFÉ two-choice assay. B–I, Quantitative data are the mean ± SEM. C, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; unexposed air versus unexposed EtOH, p = 0.3892; absorption air versus absorption EtOH, p = 0.9948; metabolism air versus metabolism EtOH, p = 0.9966. D, Left, Mann–Whitney test; air versus EtOH, ****p < 0.0001. Right, One-sample t test (theoretical mean = 0); 3 h, ****p < 0.0001; 24 h, ***p = 0.0005; 48 h, **p = 0.0090; 72 h, p = 0.2719. E, Unpaired t test; air versus EtOH, ***p = 0.0007. F, Wilcoxon signed-rank test (theoretical mean = 0); air, p = 0.6511; acute: ****p < 0.0001; chronic, ***p = 0.0007. G, Left, Welch's t test; air versus EtOH, ****p < 0.0001. Right, One-sample t test (theoretical mean = 0); repeated, ****p < 0.0001. H, Welch's t test; air versus EtOH, *p = 0.0155. I, Wilcoxon signed-rank test (theoretical mean = 0); air, p = 0.2087; acute, **p < 0.0019; repeated, *p = 0.0347.

Figure 2.

Immediate early gene induction by different tolerance paradigms. A, Quantitative PCR of transcription factor IEGs induced by acute dose, or a challenge dose following acute (rapid), chronic, or repeated exposure, expressed as the fold change versus humidified air control exposure. Left, Different ethanol pre-exposures induce different IEG response profiles. Note: kay is significantly induced when acute and chronic exposures are directly compared (p = 0.0221, Mann–Whitney test). Right, Hr38 is inducible following acute and repeated ethanol exposure conditions. B, Left, Chronic tolerance is unaffected in Hr38 mutants. Right, Chronic ethanol pre-exposure causes sedation resistance in control and Hr38 mutants. Ethanol sensitivity is unaffected in Hr38 mutants. A, B, Quantitative data are the mean ± SEM. A, Left: Hr38: Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test; acute versus rapid, ***p = 0.0008; acute versus chronic, **p = 0.0012; rapid versus chronic, p > 0.9999. Sr: Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test; acute versus rapid, p = 0.3239; acute versus chronic, **p = 0.0087; rapid versus chronic, p = 0.7261. Jra: One-way ANOVA with Tukey's multiple-comparisons test; acute versus rapid, p = 0.0961; acute versus chronic, p = 0.5183; rapid versus chronic, p = 0.5289. kay: Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test; acute versus rapid, p > 0.9999; acute versus chronic, p = 0.1467; rapid versus chronic, p = 0.0552. Right: Wilcoxon signed-rank test (theoretical mean = 1); acute, **p = 0.0039; repeated, **p = 0.0039. B, Left: Unpaired t test; control versus Hr38⁻, p = 0.0906. Right: Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; control air versus control EtOH, ****p < 0.0001; control air versus Hr38⁻ air, p = 0.8031; Hr38⁻ air versus Hr38⁻ EtOH: **p = 0.0048; control EtOH versus Hr38⁻ EtOH, p = 0.2679.

Figure 3.

Histone deacetylase inhibitors reveal deacetylation maintains encoding of chronic tolerance. A, Left, TSA, which inhibits Class I/II histone deacetylases, trends toward decreased chronic tolerance. Right, TSA does not affect ethanol sensitivity. B, Left, Nicotinamide, which inhibits NAD-dependent sirtuin class histone deacetylases, decreases chronic tolerance. Right, Nicotinamide does not affect ethanol sensitivity. C, TSA but not nicotinamide restores Hr38 ethanol inducibility by an ethanol challenge following chronic ethanol exposure, measured by quantitative PCR. D, TSA restores rapid tolerance following a chronic exposure. A–D, Quantitative data are the mean ± SEM. A, Left, Mann–Whitney test; no Rx versus TSA, p = 0.0752. Right, One-way ANOVA with Sidak's multiple-comparisons test; no Rx air versus no Rx EtOH, ***p = 0.0007; no Rx air versus TSA air, p > 0.9999; no Rx EtOH versus TSA EtOH, **p = 0.0062; TSA air versus TSA EtOH, p = 0.9249. B, Left, Unpaired t test; no Rx versus nicotinamide, *p = 0.0446. Right, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; no Rx air versus no Rx EtOH, ***p = 0.0002; no Rx air versus nicotinamide air, p = 0.3244; no Rx EtOH versus nicotinamide EtOH, **p = 0.0013; nicotinamide air versus nicotinamide EtOH, *p = 0.0180. C, No Rx: unpaired t test; air versus EtOH, **p = 0.0050; TSA: unpaired t test; air versus EtOH, p = 0.7319. nicotinamide: Mann–Whitney test; air versus EtOH, *p = 0.0499. D, No Rx: unpaired t test; air versus EtOH, *p = 0.0342; TSA: unpaired t test; air versus EtOH, p = 0.8264.

Figure 4.

Sirt1 acts in the mushroom body γ lobes to limit the expression of chronic tolerance. A, Left, Sirt1-null mutant flies develop more chronic tolerance. Right, Sirt1-null mutant flies develop less rapid tolerance. A′, Sirt1-null mutants have decreased sensitivity to ethanol sedation. B, Reduction of Sirt1 by RNAi specifically in all postmitotic neurons increases chronic tolerance. B′, Reduction of Sirt1 expression in all neurons causes decreased ethanol sensitivity. C, Reduced Sirt1 expression in all glia does not affect chronic tolerance. C′, Reduced Sirt1 expression in all glia does not affect ethanol sensitivity. D, Sirt1 RNAi in all neurons of the mushroom bodies (R13F02>Sirt1.IR), or specifically in the mushroom body γ lobes (R11D09>Sirt1.IR), increases chronic tolerance. Reduction of Sirt1 in either the mushroom body α′/β′ lobes (R35B12>Sirt1.IR) or the α/β lobes (R28H05>Sirt1.IR) does not affect chronic tolerance. D′, Reduced Sirt1 in the mushroom bodies or in each lobe causes decreased sensitivity. E, Chronic tolerance was unaffected when Sirt1 was reduced in 17d-Gal4 neurons, the site of action for Sirt1 in rapid tolerance. E′, Reduced Sirt1 in 17d-Gal4 neurons causes decreased sensitivity. A–E′, Quantitative data are the mean ± SEM. A, Left, Welch's t test; control versus Sirt1⁻, *p = 0.0162. Right, Welch's t test; control versus Sirt1⁻, **p = 0.0025. A′, Mann–Whitney test; control versus Sirt1⁻, ****p < 0.0001. B, Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test; Gal4/UAS versus Gal4, **p = 0.0097; Gal4/UAS versus UAS, ****p < 0.0001. B′, Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test, Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, ***p = 0.0004. C, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, p = 0.9243; Gal4/UAS versus UAS, p = 0.0640. C′, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, p = 0.1240. D, R13F02 panel: One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, ***p = 0.0001; Gal4/UAS versus UAS, ***p = 0.0006. R11D09 panel: Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test; Gal4/UAS versus Gal4, **p = 0.0056; Gal4/UAS versus UAS, *p = 0.0108. R35B12 panel: Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; Gal4/UAS versus Gal4, p = 0.7905; Gal4/UAS versus UAS, p = 0.4612. R28H05 panel: One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, p = 0.0913; Gal4/UAS versus UAS, **p = 0.0022. D′, R13F02 panel: One-way ANOVA with Dunnett's multiple comparisons; Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, ****p < 0.0001. R11D09 panel: One-way ANOVA with Dunnett's multiple comparisons, Gal4/UAS versus Gal4, ***p = 0.0001; Gal4/UAS versus UAS, ****p < 0.0001. R35B12 panel: One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, ****p < 0.0001. R28H05 panel: One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, ****p < 0.0001. E, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, p = 0.7108; Gal4/UAS versus UAS, p = 0.8961. E′, Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test; Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, **p = 0.0014.

Figure 5.

Chronic tolerance development is adult-specific and activity-dependent in the mushroom bodies. A, Sirt1 is required in the adult mushroom bodies for chronic tolerance development. GAL4 was suppressed by temperature-sensitive GAL80 throughout development (Dev) by rearing the flies at 18°C (GAL80 on, GAL4 blocked), and shifting them to 29°C (GAL80 off, GAL4 active) after eclosion (Adult). A 50% ethanol challenge dose was used to account for increased ethanol sedation at 29°C. A′, Adult-specific decrease of Sirt1 in the mushroom bodies did not affect sensitivity to a 50% ethanol challenge, independent of temperature during adulthood. B, Hyperpolarization of the mushroom bodies in adults with the inwardly rectifying potassium channel Kir2.1 blocks chronic tolerance development. A 50% ethanol challenge dose was used. B′, Adult-specific hyperpolarization of the mushroom bodies had no effect on sensitivity to a 50% ethanol challenge. C, Blocking synaptic vesicle release [tetanus toxin light chain (TeTx)] in the mushroom bodies in Sirt1-null mutants results in decreased chronic tolerance. C′, Blocking synaptic vesicle release in the mushroom bodies in Sirt1-null mutant flies increased ethanol sensitivity. A–C′, Quantitative data are the mean ± SEM A, Left, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, *p = 0.0416; Gal4/UAS versus UAS, **p = 0.0019. Right, One-way ANOVA with Holm–Sidak's multiple-comparisons test; Gal4/UAS versus Gal4, p = 0.9685; Gal4/UAS versus UAS, p = 0.6880. A′, Left, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, p = 0.1940; Gal4/UAS versus UAS, p = 0.0508. Right, Kruskal–Wallis ANOVA with Dunn's multiple-comparisons test; Gal4/UAS versus Gal4, *p = 0.0492; Gal4/UAS versus UAS, p = 0.6952. B, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, *p = 0.0406; Gal4/UAS versus UAS, *p = 0.0246. B′, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, **p = 0.0066; Gal4/UAS versus UAS, p = 0.2596. C, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, *p = 0.0384; Gal4/UAS versus UAS, *p = 0.0192. C′, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, **p = 0.0055.

Figure 6.

Chronic and rapid tolerance are distinct memory-like states in the brain. A, Mutants that abolish anesthesia-resistant memory (rad¹) and anesthesia-sensitive memory (amn¹) develop normal chronic tolerance at 24 h. A′, Decreased ethanol sensitivity in mutants for early forms of memory. B, Cold-shock anesthesia, blocking anesthesia-sensitive memories, does not affect chronic tolerance. B′, Increased ethanol sensitivity following cold-shock anesthesia. C, Chronic tolerance does not include 3 h anesthesia-sensitive memory-like or 3 h anesthesia-resistant memory-like states. C′, Decreased ethanol sensitivity in radish mutants, no effect of cold shock. D, Rapid tolerance is composed of 3 h anesthesia-sensitive memory-like and 3 h anesthesia-resistant memory-like states. D′, radish mutants have decreased ethanol sensitivity, as measured from the rapid tolerance inducing exposure. A–D′, Quantitative data are the mean ± SEM. A, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; control versus rad¹, p = 0.8360; control versus amn¹, p = 0.5176. A′, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; control versus rad¹, ****p < 0.0001; control versus amn¹, ****p < 0.0001. B, Unpaired t test; untreated versus cold shock, p = 0.4243. B′, Unpaired t test; untreated versus cold shock, *p = 0.0439. C, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; control versus cold shock, p = 0.0844; control versus rad¹, p = 0.5030; control versus rad¹ cold shock, p = 0.2429. C′, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; control versus cold shock, p = 0.9743; control versus rad¹, ****p < 0.0001; control versus rad¹ cold shock, ****p < 0.0001. D, One-way ANOVA with Sidak's multiple-comparisons test; control versus cold shock, ***p = 0.0007; control versus rad¹, ****p < 0.0001; rad¹ versus rad¹ cold shock, ****p < 0.0001. D′, Unpaired t test; control versus rad¹, ****p < 0.0001.

Figure 7.

Chronic ethanol creates a non-canonical LTM-like state. A, Inhibition of CREBB blocks chronic tolerance when expressed in all adult neurons (left), but not when expressed in all adult mushroom body neurons (right). A′, Adult-specific inhibition of CREBB signaling in all neurons (left), but not in mushroom body neurons (right), decreased sensitivity to a 50% ethanol challenge. B, Summary diagram of the encoding of chronic and rapid tolerance in the adult Drosophila mushroom bodies. A, A′, Quantitative data are the mean ± SEM, A, Left, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; Gal4/UAS versus Gal4, *p = 0.0114; Gal4/UAS versus UAS, *p = 0.0137. Right, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, p = 0.8458; Gal4/UAS versus UAS, p = 0.7429. A′, Left, Brown–Forsythe ANOVA with Dunnett's T3 multiple-comparisons test; Gal4/UAS versus Gal4, ****p < 0.0001; Gal4/UAS versus UAS, ****p < 0.0001. Right, One-way ANOVA with Dunnett's multiple-comparisons test; Gal4/UAS versus Gal4, **p = 0.0064; Gal4/UAS versus UAS, *p = 0.0385.