Research Paper Volume 15, Issue 4 pp 947—981

Figure 3. Male flies with downregulated dLdh expression in glia exhibit decreased long-term courtship memory with age, while flies with upregulated dLdh expression in glia exhibit decreased survival. (A) Western blot analysis of head extracts from glial transgenic male flies aged 21 days at 29°C showing elevated ectopic HA tagged dLdh expression (upper panel) and decreased endogenous dLdh levels (lower panel) in flies using a Repots-Gal4 driver to drive UAS-dLDH (with 3xHA tag) and UAS-dLdh-RNAi expression respectively (n = 3), expression was normalized to total protein content (based on Ponceau-S staining) n = 3. Each sample consisted of head extracts from 20 heads. Comparisons made by unpaired t test, ^**p < 0.01. (B) Courtship indexes for glial transgenic male flies aged 7, 14, or 21 days at 29°C post-eclosion showing courtship conditioning rejection training was decreased compared to naïve flies for all genotypes at 7 and 21 days of age, and control only at 14 days of age. n = 48–72. Naïve and trained flies were compared within each genotype at each age using one-sided Mann-Whitney U tests, ^****p < 0.0001, ^*p < 0.05. Data presented as violin plot of frequency distribution. (C) Long-term courtship memory indexes for glial transgenic trained flies represented in B. Memory was only decreased following glial dLdh downregulation at 21 days of age compared to control (H = 16.9, p = 0.0002). Genotypes at each age were compared using Kruskal-Wallis tests with Dunn’s multiple comparisons to control, ^*p < 0.05. Data presented as violin plot of frequency distribution. (D) In male flies, only glial upregulation of dLdh reduced survival compared to control and dLdh downregulation. Shaded area represents the 95% confidence interval. Curve comparison was made using a Log-rank (Mantel-Cox) test, ^****p < 0.0001. Ages chosen for memory testing are highlighted with vertical dotted lines. (E) Altered glial dLdh expression did not impact climbing ability in male flies during negative geotaxis at any age compared to control. Comparisons across age and genotype were made using a mixed-effects model with Geisser-Greenhouse correction and Tukey’s multiple comparisons within each age group. Effects of age, genotype, and age by genotype interaction are denoted on the bottom left.