Mef2 induction of the immediate early gene Hr38/Nr4a is terminated by Sirt1 to promote ethanol tolerance

Abstract

Drug naïve animals given a single dose of ethanol show changed responses to subsequent doses, including the development of ethanol tolerance and ethanol preference. These simple forms of behavioral plasticity are due in part to changes in gene expression and neuronal properties. Surprisingly little is known about how ethanol initiates changes in gene expression or what the changes do. Here we demonstrate a role in ethanol plasticity for Hr38, the sole Drosophila homolog of the mammalian Nr4a1/2/3 class of immediate early response transcription factors. Acute ethanol exposure induces transient expression of Hr38 and other immediate early neuronal activity genes. Ethanol activates the Mef2 transcriptional activator to induce Hr38, and the Sirt1 histone/protein deacetylase is required to terminate Hr38 induction. Loss of Hr38 decreases ethanol tolerance and causes precocious but short-lasting ethanol preference. Similarly, reduced Mef2 activity in all neurons or specifically in the mushroom body α/β neurons decreases ethanol tolerance; Sirt1 promotes ethanol tolerance in these same neurons. Genetically decreasing Hr38 expression levels in Sirt1 null mutants restores ethanol tolerance, demonstrating that both induction and termination of Hr38 expression are important for behavioral plasticity to proceed. These data demonstrate that Hr38 functions as an immediate early transcription factor that promotes ethanol behavioral plasticity.

Keywords: Drosophila; Hr38; Mef2; Sirt1; behavioral plasticity; ethanol tolerance; immediate early gene; mushroom bodies.

Figure 1

Hr38 is an ethanol immediate early response gene that bidirectionally regulates the development of ethanol tolerance. (A) Steady state transcript levels of genes induced by neuronal activity, 60 minutes after termination of 30 minutes ethanol or mock air exposure, presented as fold change vs no treatment (gray line) in the wild‐type control strain. One way ANOVA/Tukeys per gene, n = 5 biological replicates/condition. (B) Time course of Hr38 expression following air (black) or ethanol (blue). Data extracted from a published microarray experiment.8 (C) Time to 50% sedation (ST50) for Hr38 null mutant heterozygotes vs wild‐type controls (+/+) exposed to ethanol once (E1) for sensitivity or twice (E2‐E1) for tolerance, t‐test, n = 30 groups. (D) Ethanol sensitivity and tolerance in flies with three copies of the Hr38 genomic region (HR38.GFP) t‐test, n = 12 groups. (E) Ethanol preference in Hr38 null heterozygotes. Flies are pre‐exposed to either air or ethanol and after 16 hours placed into the 2 choice CAFÉ assay. A positive index indicates preference for ethanol intake. One‐sample t‐test compared to zero preference, n = 20 groups. (F) Hr38 null heterozygotes show avoidance of a bitter but sweeter food source in a two‐choice seeking assay. One sample t‐test compared to zero preference, n = 5 groups. (G) Ethanol accumulation immediately following a 20 minutes exposure, and after a 20 minutes rest, allowing ethanol to be metabolized; t‐test, n = 8 groups. *P < .05, **P < .01, ***P < .001

Figure 2

Hr38 protein expression in the adult brain is induced by ethanol in the mushroom bodies. (A) Cortex region of the adult fly brain expressing the Hr38.GFP fusion protein (green), showing co‐localization with the pan‐neuronal Elav nuclear protein (magenta). (B) High levels of Hr38.GFP expression in the Kenyon cell neurons (between dashed enclosures) of the mushroom body that surround the mushroom body calyx (inner dashed area) in the brain of an ethanol exposed fly. (C) Number of Hr38.GFP‐positive Kenyon cell nuclei 1.5 hours after termination of ethanol or mock air exposure. ***P < .0001, t‐test, n = 20 brains/treatment. Scale bars: 10 μm

Figure 3

Mef2 promotes ethanol induction of Hr38, and ethanol tolerance in the mushroom bodies. (A) Diagram of the Hr38 genomic locus, indicating the positions of Mef2 consensus binding sites. Consensus sites conserved in other Drosophila species are indicated by a black dot. (B) Hr38 transcript levels in untreated flies expressing dominant negative Mef2.EnR pan‐neuronally, normalized to elav‐3E1 > + (gray line). Kruskal‐Wallis/Dunn's, n = 10 biological replicates. (C) Ethanol induction of Hr38 60 minutes after ethanol treatment is blocked by Mef2.EnR. Expressed as the difference between ethanol and untreated per biological replicate. No difference was detected between genotypes for the unexposed of mock air exposures. One sample t‐test compared to 1 (no induction), n = 5. (D) Ethanol induction of Hr38 is blocked by pan‐neuronal Mef2 RNAi (Mef2.IR). One sample t‐test compared to 1, n = 6. No difference in Hr38 expression was detected between the untreated genotypes. (E) Ethanol sensitivity and tolerance with pan‐neuronal expression (elav‐c155) of Mef2 RNAi. One way ANOVA/Tukey's, n = 30 groups. (F) Ethanol sensitivity and tolerance with pan‐neuronal expression of Mef2.EnR. One way ANOVA/Tukey's, n = 14 groups. (G) Ethanol sensitivity and tolerance with expression of Mef2.EnR restricted to the mushroom body α/β neurons (17d‐Gal4). One way ANOVA/Tukey's, n = 15 to 16 groups. *P < .05, **P < .01. (H) Ethanol sensitivity and tolerance in 17d > Mef2.Enr in the presence of the mushroom body‐specific MB‐Gal80 (MB80). One way ANOVA/Tukey's, n = 10 to 12 groups. *P < .05, **P < .01

Figure 4

Sirt1 terminates Hr38 induction to promote ethanol tolerance. (A) Hr38 transcript expression levels in wild‐type controls (+/+) and Sirt1 null mutants 1 and 3 hours after treatment, normalized to untreated controls. One way ANOVA/Tukey's, n = 14 biological replicates. (B) Hr38 transcript expression levels in wild‐type control flies treated with the sirtuin end‐product inhibitor nicotinamide (70 mM). One‐sample t‐test compared to 1 (no induction), n = 5 to 10 biological replicates. (C) Sirt1 transcript levels in flies expressing dominant negative Mef2 in all neurons (elav‐3E1), normalized to elav>+. Kruskal‐Wallis/Dunn's, n = 4 to 5 biological replicates. (D) Ethanol tolerance in flies null for Sirt1 and heterozygous for Hr38. Because of the high resistance in exposure 2 in the double mutants, the time to 25% sedation (ST25) was measured. One way ANOVA/Tukey's, n = 8 to 21. (E) Hr38 transcript levels in flies mutant for Sirt1 and heterozygous for Hr38, normalized to untreated controls. One way ANOVA/Tukey's, n = 6 biological replicates. *P < .05, **P < .01