Alcohol disinhibition of behaviors in C. elegans

Abstract

Alcohol has a wide variety of effects on physiology and behavior. One of the most well-recognized behavioral effects is disinhibition, where behaviors that are normally suppressed are displayed following intoxication. A large body of evidence has shown that alcohol-induced disinhibition in humans affects attention, verbal, sexual, and locomotor behaviors. Similar behavioral disinhibition is also seen in many animal models of ethanol response, from invertebrates to mammals and primates. Here we describe several examples of disinhibition in the nematode C. elegans. The nematode displays distinct behavioral states associated with locomotion (crawling on land and swimming in water) that are mediated by dopamine. On land, animals crawl and feed freely, but these behaviors are inhibited in water. We found that additional behaviors, including a variety of escape responses are also inhibited in water. Whereas alcohol non-specifically impaired locomotion, feeding, and escape responses in worms on land, alcohol specifically disinhibited these behaviors in worms immersed in water. Loss of dopamine signaling relieved disinhibition of feeding behavior, while loss of the D1-like dopamine receptor DOP-4 impaired the ethanol-induced disinhibition of crawling. The powerful genetics and simple nervous system of C. elegans may help uncover conserved molecular mechanisms that underlie alcohol-induced disinhibition of behaviors in higher animals.

Figure 1. Crawl Behaviors Are Inhibited in Water.

Immersion in liquid results in inhibition of many behaviors in wild-type C. elegans. Notably, the feeding behavior foraging (A), spontaneous reversals (B), touch response (C), and light response (D) are all inhibited. To assess disinhibition of crawl during immersion in water, headbend frequency (E) and percent body bends with C-shape (F) were assessed. In liquid, worms exhibited only a fast, C-shaped swim. Statistical analyses comparing behaviors on land vs. water were performed using planned unpaired two-tailed t-test. Asterisks indicate P<0.001, n≥4 assays, ≥10 worms per assay for all experiments A–C, n≥15 for D–F. Error bars represent standard error of the mean.

Figure 2. Ethanol Exposure during Immersion in Liquid Results in Disinhibition of Crawl Behaviors.

Foraging (A), spontaneous reversals (B), touch response (C), and light response (D), as well as crawling kinematics (E,F) were disinhibited by EtOH. To ensure that such disinhibition was not the result of a decline in cellular function, worms treated with sodium azide were also assessed. No disinhibition was observed in these animals. EtOH treatment resulted in a reduction of bending frequency and a loss of C-shaped body posture. Animals treated with sodium azide experienced a similar decline in bending frequency, but no reduction in C-shape body posture. Statistical analyses comparing EtOH-, azide-, and untreated worms were performed using one-way ANOVA and Tukey's HSD post-hoc test or Kruskal-Wallis and Steel-Dwass-Critchlow-Fligner post-hoc test. Asterisks indicate significance in relation to untreated controls with P<0.001, n≥4 assays, ≥10 worms per assay for all experiments A–C, n≥15 for D–F. Error bars represent standard error of the mean.

Figure 3. Disinhibition of Foraging Involves Dopamine Signaling.

Loss of dopamine synthesis or D1-like dopamine signaling significantly reduced disinhibition of foraging (A). No significant reduction in disinhibition of spontaneous reversals or touch response was observed in animals lacking SLO-1, dopamine synthesis, or dopamine receptors (B,C) except for dop-4(ok1321). Although slightly varied, responses to light did not differ significantly among strains as determined by post-hoc analyses (D). Statistical analyses comparing EtOH-treated mutants to EtOH-treated WT controls were performed using one-way ANOVA and Tukey's HSD post-hoc test or Kruskal-Wallis and Steel-Dwass-Critchlow-Fligner post-hoc test. Letters indicate distinct groupings based on post-hoc statistical comparison among strains. Asterisks indicate significance in relation to WT controls (EtOH-treated or untreated, accordingly) with P<0.001, n≥4 assays, ≥10 worms per assay for all experiments. Error bars represent standard error of the mean.

Figure 4. Loss of D1-like Dopamine Receptor DOP-4 Reduces Disinhibition of Crawl.

Loss of the D1-like receptor DOP-1 resulted in a slightly lower bending frequency versus WT with EtOH treatment (A). EtOH treatment also caused uncoordination, with significantly fewer bends propagated down the animal. This phenotype was exacerbated in dop-4 mutant animals (B). Of body bends propagated down the animal, approximately half were C-shaped in most intoxicated animals, indicating disinhibition of crawl. Only animals lacking dop-4 demonstrated resistance to this effect. Statistical analyses comparing EtOH-treated mutants to EtOH-treated WT controls were performed using one-way ANOVA and Tukey's HSD post-hoc test or Kruskal-Wallis and Steel-Dwass-Critchlow-Fligner post-hoc test. Asterisks indicate significance in relation to WT controls (EtOH-treated or untreated, accordingly) with P<0.001, n≥10 worms for all experiments. Letters indicate distinct groupings based on post-hoc statistical comparison among strains. Error bars represent standard error of the mean.