Table 1.
Experimental models: C. elegans.
Table 2.
Oligonucleotides.
Fig 1.
Exposure to high doses of ethanol affects the lifespan of wild-type worms but not dop-3 and tph-1 mutants.
Survival curves of (A) wild-type (N2 strain) animals, and (B) dopamine receptor (dop-3(ok295)) and (C) serotonin synthesis (tph-1(n4622)) mutant strains from day 1 (D1) of adulthood, immediately after 24-hour treatment with 200 mM ethanol, 400 mM ethanol or no treatment. P-values were calculated by Log-rank/Mantel–Cox for each graph and indicated next to the curve: *p < 0.05, n.s.; non-significant. Log-rank/Mantel–Cox test was also performed for the comparison of each pair of conditions and only statistically significant comparisons are shown: **p < 0.01.
Fig 2.
A 24-hour ethanol exposure does not affect the swimming behavior of adult worms.
Panels plot worm locomotion data of (A) wild-type (N2 strain) animals, and (B) dopamine (dop-3(ok295)) and (C) serotonin (tph-1(n4622)) mutant strains from day 1 (D1) of adulthood, after 24 hours-treatment with 200 mM ethanol, 400 mM ethanol or no treatment, and 1 hour of post-exposure withdrawal period. Mean and standard deviation are depicted for each group. P-values were calculated by T-test corrected using the Benjamini–Hochberg false discovery rate (FDR) post hoc test (N = 3 independent experiments).
Fig 3.
Intact dopamine and serotonin neuronal circuits are required for tolerance to ethanol (A) Schematic diagram of the kinetic chemotaxis assay (“diacetyl race” paradigm).
For the “pre-exposure” period, larval 4 (L4) stage worms are subjected to no treatment or treatment with 200 mM of ethanol, or 400mM of ethanol for 24 hours (until day 1 of adulthood, D1). After the “pre-exposure” period, all the worms were removed from the ethanol infused environment to simulate a post-exposure withdrawal setting for 1 hour. After withdrawal, worms of each pre-exposure group were divided into standard “no EtOH” kinetic chemotaxis assay plates or 60 mM ethanol infused “EtOH” kinetic chemotaxis assay plates, all of them marked with a start and finish line. Performance of (A) wild-type (wild-type, N2 strain) animals, (B) dopamine (dop-3(ok295)) and (C) serotonin (tph-1(n4622)) mutant strains are depicted. P-values were calculated by 1-way ANOVA for all conditions (indicated below the legend) and by T-test for pairwise comparisons corrected using the Benjamini–Hochberg false discovery rate (FDR) post hoc test (at least N = 3 independent experiments): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The results of 2-way ANOVA are shown in S2 Table.
Fig 4.
Ethanol consumption increases dopamine and serotonin vesicle exocytosis in worm neurons.
Representative images of the proximal worm body, using day 1 (D1) adult animals of the (A) 1785[proasic-1SNB::SEpHluorin] and the (B) 3001[Ptph-1SNB::SEpHluorin] strains, treated with 200 mM ethanol, 400 mM ethanol or no treatment for 24 hours prior to imaging (40x objective lens). Mean fluorescence (a.u.; arbitrary units) and standard deviation are depicted for each group. P-values were calculated by 1-way ANOVA for all conditions (**p < 0.01 for graph [A] and non-significant for graph [B]) and by T-test for pairwise comparisons corrected using the Benjamini–Hochberg false discovery rate (FDR) post hoc test (at least N = 3 independent experiments): *p < 0.05, **p < 0.01.
Fig 5.
Dopaminergic and serotonergic circuits modulate ethanol-induced changes in stress gene transcription.
Gene expression of (A) gst-4, (B) sod-3 and (C) adh-1 in wild-type worms, and dopamine (dop-3(ok295)) and serotonin (tph-1(n4622)) mutant strains treated with 200 mM ethanol, 400 mM ethanol or no treatment for 24 hours. Standard deviation is depicted for each group. For qPCR, p-values were calculated by 1-way ANOVA for all conditions: p < 0.001 was obtained for gst-4, sod-3 and adh-1. P-values were calculated by T-test for pairwise comparisons corrected using the Benjamini–Hochberg false discovery rate (FDR) post hoc test (at least N = 6 independent experiments): *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 6.
Summary of this study.