Alcohol sensitivity and tolerance encoding in sleep regulatory circadian neurons in Drosophila

Abstract

Alcohol tolerance is a simple form of behavioural and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is a useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. We used a forward neuroanatomical screen to identify three new neural sites for rapid tolerance encoding. One of these was composed of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. We localized rapid tolerance to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as, and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link.

Keywords: Drosophila; alcohol tolerance; circadian rhythms; circuitry; sleep.

FIGURE 1

Presynaptic genes promote rapid ethanol tolerance in the mushroom body α/β lobe intrinsic neurons. (A) Scheme for induction and detection of rapid tolerance. Identical ethanol vapour exposures, E1 and E2, are separated by 4 h and result in identical accumulation and dissipation kinetics for internal ethanol concentrations. (B) Time course for ethanol sedation during continuous ethanol vapour exposure, for E1 and E2, and the calculation of ethanol tolerance. (C) Diagram of the Drosophila brain, depicting the known sites for rapid tolerance encoding. (D) Expression pattern in the mushroom body α/β neurons of the 17d-Gal4 transgene, detected by the UAS-CD8-GFP reporter transgene, and counterstained for the ELKS/CAST ortholog BRP to reveal the synaptic neuropil. (E,E′) Tolerance (E) and sensitivity (E′) when presynaptic release is blocked by expression of the tetanus toxin light chain (UAS-TeTx) in 17d-Gal4 neurons. (F,F′) Effect of decreasing expression of the Ca_v2.1 Ca²⁺ channel cac in the 17d-Gal4 pattern. (G,G′) Effect of decreasing expression of the kinase Cdk5 in the 17d-Gal4 pattern. Statistics: (E–G′) Quantitative data are mean ± SEM. (E) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: *p = 0.0129. (E′) One-way ANOVA (p = 0.0002) with Dunnett′s multiple comparisons, Gal4/UAS versus Gal4: p = 0.4929, Gal4/UAS versus UAS: **p = 0.0025. (F) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ***p = 0.0007, Gal4/UAS versus UAS: ***p = 0.0001. (F′) One-way ANOVA (p = 0.0034) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: p = 0.9781, Gal4/UAS versus UAS: **p = 0.0049. (G) Brown–Forsythe ANOVA (p = 0.0005) with Dunnett’s T3 multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: *p = 0.0394. (G′) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Here, and in subsequent figures, a dot represents an n = 1 of approximately 20 male flies.

FIGURE 2

A functional neuroanatomical screen identifies three patterns of neurons that promote rapid tolerance development through presynaptic release. (A) Tolerance difference score for 112 enhancer-Gal4 strains expressing RNAi for cac. Grey region represents 1 standard deviation from the mean of the difference scores across all tested enhancer-Gal4s. Highlighted in green are three strains that passed secondary screens and that were further characterized. (B,B′) Reduction of cac expression in R82F12-Gal4 (left), R18H11-Gal4 (middle), and R79H04-Gal4 (right) effects on rapid tolerance (B) and sensitivity (B′). (C,C′) Effect of tetanus toxin blockade of presynaptic release in the same three enhancer-Gal4 strains for rapid tolerance (C) and sensitivity (C′). (D–F) Expression pattern of the enhancer-Gal4 strains, revealed with the UAS-myr-GFP plasma membrane-tethered GFP, and counterstained with an antibody to the discs large (DLG) synaptic protein. (D′–F′) Enlargement of a substack from D–F encompassing the horizontal lobes of the mushroom bodies. Statistics: (B,C) Quantitative data are mean ± SEM. (B) Left panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: **p = 0.0040. Middle panel: One-way ANOVA (p = 0.0293) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: *p = 0.0376, Gal4/UAS versus UAS: *p = 0.0379. Right panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: *p = 0.0162. (B′) Left panel: Kruskal–Wallis test (p = 0.0001) with Dunn’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: **p = 0.0059. Middle panel: One-way ANOVA (p = 0.1120). Right panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: *p = 0.0126. (C) Left panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ***p = 0.0003, Gal4/UAS versus UAS: ****p < 0.0001. Middle panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ***p = 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Right panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. (C′) Left panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: p = 0.8707, Gal4/UAS versus UAS: ****p < 0.0001. Middle panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Right panel: Kruskal–Wallis test (p < 0.0001) with Dunn’s multiple comparisons, Gal4/UAS versus Gal4: ***p = 0.0007, Gal4/UAS versus UAS: ***p = 0.0001.

FIGURE 3

Distinct rapid tolerance neurons exist in each enhancer-Gal4, including glutamatergic dorsal clock neurons in R18H11-Gal4. (A,A′) Vesicular glutamate transporter vGlut RNAi effects on rapid tolerance (A) and sensitivity (A′). (B,B′) Reduction of vGlut expression using a second independent RNAi transgene, effects on tolerance (B) and sensitivity (B′) in R82F12-Gal4 expressing neurons. (C,C′) Adult specific vGlut RNAi in R82F12-Gal4 expressing neurons, effect on rapid tolerance (C) and sensitivity (C′). (D,D′) Counterstaining of R82F12>myr-GFP brains with anti-CCHa1 to detect DN1a neurons. (E,E′) Counterstaining of R82F12>myr-GFP brains with anti-DH31 to detect DN1p neurons. Micrographs in D and E depict the dorsal hemisphere of an adult brain. (F,F′) Split-Gal4 driving expression of vGlut RNAi in neurons that are common between R82F12 and R18H11, effect on rapid tolerance (F) and sensitivity (F′). (G) Split-Gal4 expression pattern revealed with UAS-myr-GFP. (H) Effect of vGlut RNAi in R18H11-Gal4 neurons on ethanol absorption and metabolism. Genetic background is the w- Berlin strain used to outcross all genetic reagents. Statistics: (A–C,F) Quantitative data are mean ± SEM. (A) Left panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Middle panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Right panel: One-way ANOVA (p = 0.0588). (A′) Left panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Middle panel: Brown–Forsythe ANOVA (p < 0.0001) with Dunnett’s T3 multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Right panel: Kruskal–Wallis test (p = 0.0038) with Dunn’s multiple comparisons, Gal4/UAS versus Gal4: *p = 0.0141, Gal4/UAS versus UAS: **p = 0.0038. (B) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: *p = 0.0398, Gal4/UAS versus UAS: ****p < 0.0001. (B′) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. (C) One-way ANOVA (p = 0.0018) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: *p = 0.0209, Gal4/UAS versus UAS: **p = 0.0012. (C′) Brown–Forsythe ANOVA (p = 0.0078) with Dunnett’s T3 multiple comparisons, Gal4/UAS versus Gal4: **p = 0.0090, Gal4/UAS versus UAS: p = 0.1720. (F) One-way ANOVA (p = 0.0002) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: **p = 0.0038, Gal4/UAS versus UAS: p = 0.2004. (F′) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. (H) One-way ANOVA (p = 0.0067) with šídák’s multiple comparisons, no treatment: p = 0.9716, absorption: p = 0.9423, metabolism: p = 0.7767.

FIGURE 4

DN1a and downstream evening circadian neurons promote rapid tolerance. (A) Presence (+) and absence (−) of expression of enhancer-Gal4s and neurotransmitter/neuromodulators in the DN1a and DN1p dorsal clock neurons. (B,B′) Reduction of glutamate release with vesicular glutamate transporter vGlut RNAi in enhancer-Gal4 patterns that include one or both of the dorsal group clock neurons, effects on rapid tolerance (B) and sensitivity (B′). (C) Ten presynaptic (left) and postsynaptic (right) neurons that make the greatest number of synapses with the DN1a neurons, expressed as the sum total for the right pair of DN1a neurons in the hemibrain electron microscopy reconstruction. The diagram below depicts the morphology of one of the pair of DN1a neurons and the LNd6 and 5th LNv postsynaptic neurons in the full adult female brain (FAFB) electron microscopy reconstruction. SMP: superior medial protocerebrum; LHPV/LHAV: lateral horn posterior/anterior ventral; aMe: accessory medulla. DvPdf-Gal4 is expressed in the LNd6 and 5th LNv neurons. (D,D′) Reduction of metabotropic glutamate receptor mGluR expression in DvPdf-Gal4 expressing neurons, effect on rapid tolerance (D) and sensitivity (D′). (E,E′) Reduction of PDF receptor Pdfr expression in R18H11-Gal4 expressing neurons, effect on rapid tolerance (E) and sensitivity (E′). Statistics: (B,B′,D–E′) Quantitative data are mean ± SEM. (B) Left panel: One-way ANOVA (p = 0.0013) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ***p = 0.0007, Gal4/UAS versus UAS: *p = 0.0493. Middle panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: *p = 0.0195. Right panel: One-way ANOVA (p = 0.0002) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: **p = 0.0018, Gal4/UAS versus UAS: p = 0.8003. (B′) Left panel: One-way ANOVA (p = 0.0561). Middle panel: One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ****p < 0.0001. Right panel: Brown–Forsythe ANOVA (p < 0.0001) with Dunnett’s T3 multiple comparisons, Gal4/UAS versus Gal4: ***p = 0.0009, Gal4/UAS versus UAS: ****p < 0.0001. (D) Brown–Forsythe ANOVA (p < 0.0001) with Dunnett’s T3 multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: ***p = 0.0005. (D′) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: *p = 0.0221. (E) Brown–Forsythe ANOVA (p = 0.5724). (E′) One-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons, Gal4/UAS versus Gal4: ****p < 0.0001, Gal4/UAS versus UAS: p = 0.9985.