Alcohol Causes Lasting Differential Transcription in Mushroom Body Neurons.

Exposure paradigms and differentially expressed genes in MB nuclei. (A) Paradigm depicting three spaced 10 min exposures of “Air”, “Ethanol”, “Odors”, or “Trained” (odor + ethanol) and flies killed 24 hr later. RNA for each biological replicate (n) was extracted from mushroom body nuclei isolated from ∼2000 male heads [Air: n = 3, Ethanol: n = 3, Odors: n = 4 (two reciprocals of opposite odor order), Trained: n = 4 (two reciprocals of opposite odor order)]. (B and C) Volcano plots showing fold change of gene expression [log2(fc+1)] compared to the inverse of statistical significance [−log10(P-value)] (dark outline, FDR < 0.05). Both (B) Air vs. Odors and (C) Odors vs. EtOH had one statistically significant differentially expressed gene. (D and E) Density histogram and normalized FPKM expression (blue, yellow, and red color representing relative levels) of (D) the 100 most variable genes—the squared deviation from the mean—and (E) the 20 highest expressed genes postfiltering of RNA-associated genes. FDR, false discovery rate.

Comparison of differential transcripts in response to odor, ethanol, or trained (odor-ethanol) treatment. (A) Volcano plots showing fold change of transcript expression [log2(fc+1)] compared to the inverse of statistical significance [−log10(P-value)] (dark outline, FDR < 0.05). Plots for (i) Odors, (ii) EtOH, and (iii) Trained (ethanol-odor pairing) compared to Air (colors depict the top 200 P-value transcripts). (B) An upgraded Venn Diagram plot generated by an R package called “UpSetR” demonstrating the intersection in top 200 P-value transcripts (abbreviated by first letter in treatment). (C) A few of the transcripts from intersectional analysis are listed, along with the corresponding highest DIOPT-scored human genes. Hypergeometric statistics, *P < 0.05 (Figure S4C). FDR, false discovery rate.

Differential transcripts in particular pairwise treatment comparisons. (Ai–Ci) Volcano plots depicting (A) how EtOH differentially affects transcript expression compared to odors, (B) how Training (ethanol-odor pairing) differentially affects transcript expression compared to EtOH alone, and (C) how Training (ethanol-odor pairing) differentially affects transcript expression compared to Odors alone (dark outline, FDR < 0.05). (Aii–Cii) Significantly altered transcripts are depicted alongside the most highly expressed isoform of the same gene, gray shaded regions denote sequence differences between isoforms. (Aiii–Ciii) Transcripts are listed, along with the corresponding highest DIOPT-scored human genes. FDR, false discovery rate.

Protein interaction network analysis of alternatively expressed transcripts. Protein interaction network of proteins associated with “Odor vs. Trained” significant transcripts. Each node represents a protein, dark outlines denote proteins associated with significant “Odor vs. Trained” transcripts. Attributes of the nodes include fold change [log2(fc+1)] as color, expression level as size [log2(FKPM+1)] and edge thickness between nodes represents the extent of protein-protein interaction evidence from the MIST database.

Spliceosome complex gene Cdc5 is required for cue-induced ethanol memory in adult MB neurons. (A) A Cdc5-GFP fusion shows that Cdc5 is expressed diffusely in the (i) anterior and (ii) posterior adult central brain, including (iii and iv) within MB nuclei (red). Bar in i, ii, and iii is 50 µM, Bar in iv is 5 µM). (B) Expression of Cdc5-RNAi in adult MB neurons significantly reduced conditioned preference for odor-cue-induced ethanol memory [F(2,56) = 21, *P < 0.0001]. Expression pattern of R19B03-Gal4 is shown in Figure S9A, and efficacy of the RNAi in Figure S10. (C) Primers designed to measure expression of transcripts that include the Stat92E–RH (Exon 1) or –RI (Exon 1a) isoforms (Henriksen et al. 2002), see Figure S8A for depiction of all Stat92E transcripts. (D) Decreasing expression of Cdc5 in adult neurons significantly alters the ratio of Exon1/1a transcripts in whole-head tissue [F(2,14) = 5.7, *P = 0.02].

Knockdown of splice-associated targets in MB neurons reduces odor-cue-induced ethanol memory. (A) Density histogram and normalized FPKM expression (blue, yellow, and red color representing relative levels) of genes associated with the spliceosome; arrows mark selected candidate targets for testing. (B) Table listing four top-expressed spliceosome-associated genes and Cdc5, along with their corresponding highest DIOPT-scored human genes (Herold et al. 2009; Berson et al. 2019), and schematic demonstrating the complexes associated with these genes during splicing. (C) Expression of RNAi targeting Rm62 [F(2,49) = 5.72, P = 0.02], CG7971 [F(2,55) = 3.75, P = 0.03], Ref1 [F(2,44) = 6.96, P = 0.002], or Pep [F(2,43) = 9.30, P = 0.0004] in adult MB neurons significantly reduced conditioned preference for odor-cue-induced ethanol memory.