Memory-like states created by the first ethanol experience are encoded into the Drosophila mushroom body learning and memory circuitry in an ethanol-specific manner

Abstract

A first ethanol exposure creates three memory-like states in Drosophila. Ethanol memory-like states appear genetically and behaviorally paralleled to the canonical learning and memory traces anesthesia-sensitive, anesthesia-resistant, and long-term memory ASM, ARM, and LTM. It is unknown if these ethanol memory-like states are also encoded by the canonical learning and memory circuitry that is centered on the mushroom bodies. We show that the three ethanol memory-like states, anesthesia-sensitive tolerance (AST) and anesthesia resistant tolerance (ART) created by ethanol sedation to a moderately high ethanol exposure, and chronic tolerance created by a longer low concentration ethanol exposure, each engage the mushroom body circuitry differently. Moreover, critical encoding steps for ethanol memory-like states reside outside the mushroom body circuitry, and within the mushroom body circuitry they are markedly distinct from classical memory traces. Thus, the first ethanol exposure creates distinct memory-like states in ethanol-specific circuits and impacts the function of learning and memory circuitry in ways that might influence the formation and retention of other memories.

Fig 1. An acute, sedating dose of ethanol forms separable AST and ART.

A) Cold shock anesthesia decreases 4 hr but not 24 hr rapid tolerance. Left and center-left: Rapid tolerance is cold shock sensitive for at least 3 hr after acute ethanol exposure. Center-right and right: rapid tolerance measured at 24 hr is cold-shock insensitive. Unpaired t test (two-tailed). B) amn promotes AST and sedation sensitivity, but not ART. Left: unpaired t test (two-tailed); right: Welch’s t test (two-tailed). Sensitivity: Welch’s t test (two-tailed). C) Neuronal amn promotes AST and sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s. Sensitivity, right: one-way ANOVA/Holm-Šídák’s. D) Neuronal rsh supports ART and sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s. Sensitivity: one-way ANOVA/Holm-Šídák’s. E) Neuronally encoded AST and ART dynamics for rapid tolerance encoding.

Fig 2. Amnesiac-dependent AST resides in the mushroom body Kenyon cells.

A, A’) amn in all mushroom body Kenyon cells promotes rapid tolerance and its effect is sensitive to cold shock, whereas there is no effect on sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; right: one-way ANOVA. Sensitivity, left: one-way ANOVA; right: Kruskal-Wallis. B, B’) amn is dispensable in DPM neurons for rapid tolerance and sedation sensitivity. Tolerance: one-way ANOVA. Sensitivity: Kruskal-Wallis. C, C’) amn in the mushroom body γ lobe Kenyon cells promotes rapid tolerance. amn in the mushroom body αβ lobe Kenyon cells promotes sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; center: one-way ANOVA; right: one-way ANOVA. Sensitivity, left: Kruskal-Wallis/Dunn’s; center: one-way ANOVA; right: one-way ANOVA/Holm-Šídák’s. D, D’) Nmdar1 in all mushroom body Kenyon cells promotes AST and sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; right: one-way ANOVA. Sensitivity: one-way ANOVA/Holm-Šídák’s. E) Summary of AST encoding by anesthesia, NMDAR, and amn in the mushroom body circuitry, as compared to ASM.

Fig 3. Radish-dependent ART resides outside the mushroom bodies.

A, A’) rsh in the Kenyon cells is dispensable for ART, whereas it promotes sedation sensitivity. Tolerance, left: Brown-Forsythe ANOVA; center: Kruskal-Wallis; right: one-way ANOVA. Sensitivity: one-way ANOVA/Holm-Šídák’s. B, B’) rsh in the APL neurons is dispensable for ART, but it promotes sedation sensitivity. Tolerance: Brown-Forsythe. Sensitivity: one-way ANOVA/Holm-Šídák’s. C, C’) rsh in the DPM neurons is dispensable for rapid tolerance and sedation sensitivity (C’). Tolerance: one-way ANOVA/Holm-Šídák’s. Sensitivity: one-way ANOVA. D) Radish-dependent encoding of ART, as compared to ARM, in the mushroom body circuitry.

Fig 4. APL activity during tolerance acquisition promotes rapid tolerance.

A, A’) Left: inactivation of the APL neurons throughout the rapid tolerance paradigm decreased rapid tolerance (A), and increased sedation sensitivity (A’). Right: subtraction of GABAergic neurons (Gad1-Gal80), including the APL, blocks the effects of neuronal inactivation for both rapid tolerance and sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; right: Kruskal-Wallis. Sensitivity, left: Brown-Forsythe/Dunnett’s; right: Kruskal-Wallis/Dunn’s. B, B’) Inactivation of the APL neurons in GH146-Gal4 specifically during tolerance acquisition decreased rapid tolerance (B). Inactivation of neurons in the GH146-Gal4 pattern during the inter-exposure interval or during the expression phase increased sedation sensitivity (B’). Tolerance, left: one-way ANOVA/Holm-Šídák’s; center: one-way ANOVA; right: Brown-Forsythe. Sensitivity, left: one-way ANOVA; center: one-way ANOVA/Holm-Šídák’s; right: one-way ANOVA/Holm-Šídák’s. C, C’) APL inactivation during rapid tolerance acquisition decreased ART measured at 24 hr. Tolerance: one-way ANOVA/Holm-Šídák’s. Sensitivity: one-way ANOVA. D, D’) RNAi against Gad1 in the APL neurons increases rapid tolerance (left), and the effect is sensitive to cold shock (right). There is no effect on sedation sensitivity. Tolerance, left: Brown-Forsythe/Dunnett’s; right: one-way ANOVA. Sensitivity: one-way ANOVA. E, E’) RNAi against Tbh in the APL neurons increases rapid tolerance (left), and the effect is sensitive to cold shock (right). There is no effect on sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; right: one-way ANOVA/Holm-Šídák’s. Sensitivity: one-way ANOVA. F) Activity-dependent encoding of AST and ART, as compared to ASM and ARM.

Fig 5. AST requires GABAergic repression of the Kenyon cells, but not ongoing neuronal activity.

A, A’) GABA_A receptor subunit Rdl is required to promote AST in mushroom body Kenyon cells, with no effect on sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; right: one-way ANOVA. Sensitivity: one-way ANOVA/Holm-Šídák’s. B, B’) Neuronal activity is not required in the mushroom body Kenyon cells for rapid tolerance or sedation sensitivity. Tolerance, left: Kruskal-Wallis; center-left: one-way ANOVA; center-right: one-way ANOVA; right: one-way ANOVA. Sensitivity, left: Kruskal-Wallis; center-left: one-way ANOVA; center-right: one-way ANOVA/Holm-Šídák’s; right: one-way ANOVA. C) Role of GABAergic signaling and neuronal activity in the mushroom body Kenyon cells for AST and ASM.

Fig 6. Chronic tolerance requires protein synthesis in adult neurons but not in the mushroom body Kenyon cells.

A, A’) Protein synthesis in neurons is required during the chronic tolerance paradigm. No effect on sedation sensitivity. Tolerance: one-way ANOVA/Holm-Šídák’s. Sensitivity: one-way ANOVA/Holm-Šídák’s. B, B’) Protein synthesis is not required in the mushroom body Kenyon cells for chronic tolerance or for sedation sensitivity. Tolerance, left and center: one-way ANOVA; right Brown-Forsythe. Sensitivity, left: One-way ANOVA/Holm-Šídák’s; center: Brown-Forsythe; right: one-way ANOVA. C, C’) Left: dominant negative Mef2 in the mushroom body Kenyon cells does not affect chronic tolerance or sedation sensitivity. One-way ANOVA. Middle: Nmdar1 is not required in the mushroom body Kenyon cells for chronic tolerance. It promotes sedation sensitivity there. Tolerance: one-way ANOVA. Sensitivity: one-way ANOVA/Holm-Šídák’s. Right: constitutively active CaMKII in the mushroom body Kenyon cells does not affect chronic tolerance or sedation sensitivity. Tolerance: one-way ANOVA. Sensitivity: one-way ANOVA/Holm-Šídák’s. D, D’) The rut adenylyl cyclase is required for chronic tolerance and sedation sensitivity. Tolerance: Mann-Whitney test (two-tailed). Sensitivity: unpaired t test (two-tailed). E) Chronic tolerance molecular encoding mechanisms occur outside the mushroom body circuitry, in contrast to classical LTM.

Fig 7. DPM synaptic release is required during chronic ethanol exposure for chronic tolerance.

A, A’) DPM synaptic release is required during the chronic paradigm for chronic tolerance development, and it is not required for sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; right: one-way ANOVA. Sensitivity, left: Brown-Forsythe/Dunnett’s; right: Kruskal-Wallis. B, B’) DPM synaptic release is specifically required during chronic ethanol exposure for tolerance development. Tolerance, left: one-way ANOVA/Holm-Šídák’s; center and right: one-way ANOVA. Sensitivity, left: Brown-Forsythe/Dunnett’s; center: Brown-Forsythe; right: one-way ANOVA. Sensitivity, left: Brown-Forsythe/Dunnett’s; center: Brown-Forsythe; right: one-way ANOVA. C, C’) APL synaptic release is dispensable for chronic tolerance and for sedation sensitivity. Tolerance: one-way ANOVA. Sensitivity: Kruskal-Wallis. D, D’) Mushroom body Kenyon cell synaptic release, tested in each lobe, is dispensable for chronic tolerance. Synaptic release in the αβ Kenyon cells is required for ethanol sensitivity. Tolerance, left: one-way ANOVA; center-left: Kruskal-Wallis/Dunn’s; center-right: Brown-Forsythe; right: Brown-Forsythe/Dunnett’s. Sensitivity left: Brown-Forsythe/Dunnett’s; center-left: Kruskal-Wallis; center-right: Brown-Forsythe/Dunnett’s; right: one-way ANOVA/Holm-Šídák’s. E) Role of synaptic release in the mushroom body circuitry for chronic tolerance and long-term memory.

Fig 8. DPM GABAergic repression of mushroom body Kenyon cells is compartmentalized to the αβ lobes to promote chronic tolerance.

A, A’) GABA synthesis is required in the DPM neurons to promote chronic tolerance and sedation sensitivity. Tolerance, left: one-way ANOVA/Holm-Šídák’s; right: Kruskal-Wallis/Dunn’s. Sensitivity, left: one-way ANOVA/Holm-Šídák’s; right: Kruskal-Wallis/Dunn’s. B, B’) Rdl in the adult mushroom body Kenyon cells promotes chronic tolerance, but not sedation sensitivity. Tolerance: Kruskal-Wallis/Dunn’s. Sensitivity: one-way ANOVA. C, C’) Rdl in the αβ Kenyon cells promotes chronic tolerance and sedation sensitivity. Tolerance, left and center: one-way ANOVA; right: one-way ANOVA/Šídák’s. Sensitivity, left, center, and right: one-way ANOVA/Šídák’s. D) Summary comparison of DPM roles in chronic tolerance and long-term memory.

Fig 9. DPM neurons require protein synthesis, but not CREB, to promote chronic tolerance.

A, A’) DPM neurons require protein synthesis during the chronic exposure, but they do not depend on CREB for chronic tolerance. Sedation sensitivity is unaffected. Tolerance, left: Brown-Forsythe; middle: Kruskal-Wallis/Dunn’s; right: one-way ANOVA. Sensitivity, left: Kruskal-Wallis; middle: one-way ANOVA/Šídák’s; right: Brown-Forsythe. B, B’) Neither protein synthesis nor the kayak immediate early gene are required in the mushroom body αβ Kenyon cells for chronic tolerance or sedation sensitivity. Tolerance, left: Brown-Forsythe; all others: one-way ANOVA. Sensitivity, left: Brown-Forsythe; center-left: Kruskal-Wallis/Dunn’s; center-right: one-way ANOVA; right: Brown-Forsythe/Dunnett’s. C) Summary diagram of protein synthesis dependence in the mushroom bodies for chronic tolerance and LTM.

Fig 10. Sirt1 suppresses formation of a labile trace in the mushroom body

γ lobe Kenyon cells that consolidates into enhanced chronic tolerance. A, A’) Sirt1 null mutants exhibit higher chronic tolerance that is anesthesia sensitive early but not late during the inter-exposure interval. Tolerance, left: unpaired t test (two-tailed); middle: unpaired t test (two-tailed); right: Mann-Whitney test (two-tailed). Sensitivity, left: Mann-Whitney test (two-tailed); middle: Mann-Whitney test (two-tailed); right: unpaired t test (two-tailed). B, B’) Labile chronic tolerance trace localizes to the mushroom body γ lobes. Tolerance: one-way ANOVA. Sensitivity: one-way ANOVA/Holm-Šídák’s. C, C’) Sirt1 null mutant enhanced chronic tolerance is dissipated by 72 hr. Tolerance, left: one-sample t test, compared to zero; right: one-way ANOVA. Sensitivity, left: unpaired t test (two-tailed); right: one-way ANOVA. D) No Sirt1 null-dependent enhancement of rapid tolerance in the mushroom body γ lobes. Left: Brown Forsythe; right: one-way ANOVA. E) Model for Sirt1-suppression of a labile trace that consolidates to enhance chronic tolerance.

Fig 11. Summary diagrams of mushroom body genes and circuits for Drosophila ethanol behaviors and classical forms of associative memory.

Blue (rapid) and green (chronic) boxes contain pathways for ethanol tolerance and the orange box for ethanol sensitivity. Gray boxes contain pathways for associative memory that were tested for ethanol behavior.