Alcohol sedation in adult Drosophila is regulated by Cysteine proteinase-1 in cortex glia

Abstract

Although numerous studies have demonstrated that neuronal mechanisms regulate alcohol-related behaviors, very few have investigated the direct role of glia in behavioral responses to alcohol. The results described here begin to fill this gap in the alcohol behavior and gliobiology fields. Since Drosophila exhibit conserved behavioral responses to alcohol and their CNS glia are similar to mammalian CNS glia, we used Drosophila to begin exploring the role of glia in alcohol behavior. We found that knockdown of Cysteine proteinase-1 (Cp1) in glia increased Drosophila alcohol sedation and that this effect was specific to cortex glia and adulthood. These data implicate Cp1 and cortex glia in alcohol-related behaviors. Cortex glia are functionally homologous to mammalian astrocytes and Cp1 is orthologous to mammalian Cathepsin L. Our studies raise the possibility that cathepsins may influence behavioral responses to alcohol in mammals via roles in astrocytes.

Keywords: Behavioural genetics; Glial biology.

Fig. 1

Cp1 knockdown in CNS glia alters ethanol sedation sensitivity without affecting internal ethanol levels. a, b ST50 values were reduced in flies expressing Cp1 RNAi transgenes in glia (blue bars: repo-Gal4/v13959, panel a; repo-Gal4/HMS00725, panel b) compared to control flies with either repo-Gal4 alone (black bars: repo-Gal4/+) or the RNAi transgenes alone (black bars: v13959/+ and HMS00725/+) (panel a: one-way ANOVA, p = 0.0352; *Bonferroni’s multiple comparison vs. controls, p < 0.05; n = 8; panel b: one-way ANOVA, p < 0.0001; *Bonferroni’s multiple comparison vs. control, p < 0.05; n = 8). c, d Expression of Cp1 RNAi transgenes in CNS glia (blue bars: v13959, panel c; HMS00725, panel d) did not alter internal ethanol levels compared to controls with either repo-Gal4 or the RNAi transgenes alone (black bars) (individual one-way ANOVAs, p > 0.05; n = 8). e–h Whole-mount brain images immunolabeled for Cp1 expression (n = 5). Whole-brain Cp1 detection was reduced in flies expressing Cp1 RNAi transgenes in glia (f, h) compared to brains from RNAi transgene control animals (e, g). (Anti-Cp1 1:250, Alexa 568 1:1000). Representative images, ×10

Fig. 2

Trans-species rescue of alcohol sedation in Cp1 RNAi flies. a, c Ethanol sedation in flies with repo-Gal4 alone, HMS00725 alone, UAS-GA25021 transgenes alone, and repo-Gal4 with UAS-GA25021. Genotype did not impact ST50 values (panel a: one-way ANOVA, p = 0.4855, n = 8; panel c: one-way ANOVA, p = 0.1683, n = 8). b, d Ethanol sedation in flies with concurrent expression of Cp1 RNAi and GA25021. ST50 values were decreased in flies constitutively expressing the HMS00725 Cp1 RNAi transgene in all glia via repo-Gal4 (blue squares) compared to control flies containing repo-Gal4 alone (black circles). ST50 values in flies that expressed a UAS-GA25021 transgene and HMS00725 Cp1 RNAi in all glia via repo-Gal4 (gray triangles: UAS-GA25021 #1, panel b; UAS-GA25021 #3, panel d) were significantly elevated compared to flies expressing HMS00725 alone (blue squares: UAS-GA25021 #1, panel b; UAS-GA25021 #3, panel d), but were not different than control flies containing repo-Gal4 alone (black circles) (panel b: one-way ANOVA, p < 0.0001, n = 8,; panel d: one-way ANOVA, p = 0.0019; *Bonferroni’s multiple comparison vs. repo-Gal4;HMS00725 flies, p < 0.05). e–j Whole-mount brain images immunolabeled for Cp1. Whole-brain fluorescence was reduced in flies constitutively expressing the HMS00725 Cp1 RNAi transgene in all glia via repo-Gal4 (f) compared to brains that contained repo-Gal4 alone (e). Compared to brains that contained repo-Gal4 alone (e), whole-brain fluorescence was increased when a UAS-GA25021 transgene was expressed in all glia via repo-Gal4 (UAS-GA25021 #1, panel g; UAS-GA25021 #3, panel i). Compared to brains that expressed the HMS00725 Cp1 RNAi transgene in all glia via repo-Gal4 (f), whole-brain fluorescence was increased when a UAS-GA25021 transgene was expressed with the HMS00725 Cp1 RNAi transgene in all glia via repo-Gal4 (UAS-GA25021 #1, panel h; UAS-GA25021 #3, panel j). Representative images from middle sections of adult brains, ×10 (Anti-Cp1 1:250; Alexa 568 1:1000)

Fig. 3

Cp1 expression in cortex glia is required for normal ethanol sedation. a, b ST50 values were decreased in flies expressing Cp1 RNAi transgenes in cortex glia (blue bars: NP2222-Gal4/v13959, panel a; NP2222-Gal4/HMS00725, panel b) compared to control flies containing either the cortex glia Gal4 driver (black bars: NP2222-Gal4/+) or the RNAi transgenes (black bars: v13959/+ or HMS00725/+) alone (individual one-way ANOVAs, p ≤ 0.0001; *Bonferroni’s multiple comparisons vs. controls, p < 0.05; n = 8). c–h Cp1 is expressed in cortex glia. c, d Whole-brain expression of UAS-GFP (green) driven by NP2222. e, f Endogenous Cp1 expression labeled red (anti-Cp1 1:250, Alexa 568 1:1000). g, h Merged image of panels c and e (g), and panels d and f (h); GFP and Cp1 co-localization is yellow. Representative images from whole brain at ×10 (c, e, g) and 63x oil-immersion (d, f, h)

Fig. 4

Cp1 in rapid tolerance development. a ST50 values from the first (E1) and second (E2) ethanol exposure when Cp1 is knocked down in all CNS glia. Compared to controls (repo-Gal4/+ and v13959/+), expression of Cp1 RNAi in CNS glia (repo-Gal4/v13959) decreased ST50 values during E1, but not during E2 (two-way ANOVA; genotype, n.s.; ethanol exposure, p < 0.0001; interaction, p = 0.015; *Bonferroni’s multiple comparisons vs. controls for each ethanol exposure, p < 0.05; n = 8). b Development of rapid tolerance (fold change in ST50 from E1 to E2) quantified from the data in a. Expression of Cp1 RNAi in glia (blue bar: repo-Gal4/v13959) increased rapid tolerance development compared to controls (black bars: repo-Gal4/+, v13959/+) (one-way ANOVA, p = 0.0014; *Bonferroni’s multiple comparisons vs. controls, p < 0.05; n = 8). c ST50 values from the first (E1) and second (E2) ethanol exposure when Cp1 is knocked down in cortex glia. Compared to controls (NP2222-Gal4/+ and v13959/+), expression of Cp1 RNAi in cortex glia (NP2222-Gal4/v13959) decreased ST50 during E1, but not during E2 (two-way ANOVA; ethanol exposure, p < 0.0001; genotype, p = 0.0034; interaction, p = 0.0001; *Bonferroni’s multiple comparisons vs. controls for each ethanol exposure, p < 0.05; n = 8). d Development of rapid tolerance (fold change in ST50 from E1 to E2) quantified from the data in c. Expression of Cp1 RNAi in cortex glia (blue bar: NP2222-Gal4/v13959) increased rapid tolerance development compared to controls (black bars: NP2222-Gal4/+, v13959/+) (one-way ANOVA, p = 0.0009; *Bonferroni’s multiple comparisons vs. controls, p < 0.05; n = 8)

Fig. 5

Cp1 knockdown in CNS glia during adulthood increased ethanol sedation sensitivity. a, b Compared to vehicle, treatment with 1 mM RU486 for 6 days decreased ST50 values in flies with the GliaGS driver and a Cp1 RNAi transgene (GliaGS/v13959, panel a; GliaGS/HMS00725, panel b), but not in control flies with either GliaGS or an RNAi transgene alone (panel a: two-way ANOVA; RU486, p = 0.0247; genotype, n.s.; interaction, n.s.; *Bonferroni’s multiple comparisons between vehicle and RU486, p < 0.05; n = 8; panel b: two-way ANOVA; RU486, n.s.; genotype, n.s.; interaction, p = 0.0411; *Bonferroni’s multiple comparisons between vehicle and RU486, p < 0.05; n = 16). c–h GliaGS drives expression in CNS glia during adulthood. GliaGS/LacZ flies were fed 1 mM RU486 for 6 days prior to brain dissection and immunolabeling. c, d Endogenous repo expression (green) indicating CNS glia (anti-repo 1:100, Alexa 488 1:1000) (e, f) GliaGS-driven LacZ expression labeled red (anti-LacZ 1:500, Alexa 568 1:1000) (g, h) merged images of panels c and e (g) and panels d and f (h); yellow indicates co-localization of repo and LacZ. Representative images from ×10, scale bar = 100 µm (c, e, g) and 63x oil, scale bar = 10 µm (d, f, h). i Treatment of GliaGS/UAS-LacZ flies with 1 mM RU486 for 6 days induced β-galactosidase activity in whole-fly extracts (blue line) compared to vehicle control (black line)