Figure 4. RNAi knock down of G_αq impairs forgetting.

A. The graphic shows the structure of the Gαq gene with two annotated isoforms, G and D. Two different transcriptional units and alternative splicing encode these forms. The open reading frame is illustrated as solid black with 5’ and 3’ untranslated regions colored light yellow. Three different RNAi lines were used in this study. All three Gαq RNAi transgenes encode short hairpin RNAi sequences complementary to 24 nucleotides of two annotated isoforms, G and D, with RNAi2 and 3 targeting the 5’ UTR in exon 4 and Gαq RNAi1 targeting sequences in exon 10. Supplementary Figure 3 illustrates an expanded exon 4 and 10 and the specific sequences targeted by the three RNAi lines.

B. RNAi knock down of Gαq using the pan-neuronal c155-gal4 driver and tub-gal80^ts to control expression only during adulthood with a temperature shift from 18°C to 30°C after eclosion. The tub-gal80^ts transgene expresses temperature-sensitive Gal80^ts protein that inhibits Gal4 activity at 18°C but not at 30°C. The tubulin (tub) promoter drives expression of gal80^ts in all cells. Knockdown of Gαq with RNAi2 increased memory expression relative to the “uas-control”. The “uas-control” is a chromosome containing the attP40 docking site used for the insertion of the RNAi constructs. Statistics: Results are expressed as the mean ± SEM. Two-tailed, two-sample Student’s t-test, *p<0.05, n=13 per group.

C. RNAi knock down of Gαq expression in the MBn using the R13-gal4 driver only during adulthood increased memory expression. Statistics: Results are expressed as the mean ± SEM. Two-tailed, two-sample Student’s t-test, *p<0.05, **p<0.001, n=12–16 per group.

D. RNAi knock down of Gαq expression only during adulthood with two additional RNAi transgenes increased memory performance. Statistics: Results are expressed as the mean ± SEM. Two-tailed, two-sample Student’s t-test, *p<0.05, n=12 per group.

E. Memory retention was enhanced with Gαq-RNAi expression in the MBn only at intermediate time – 3 and 4.5 hr – after conditioning. Statistics: Results are expressed as the mean ± SEM. Two-tailed, two-sample Student’s t-test, *p<0.05, **p<0.01, n=8–12 per group.

F. RNAi knock down of Gαq expression in adult MBn did not alter the memory acquisition curve with flies trained with an increasing number of electric shock pulses. Statistics: Results are expressed as the mean ± SEM. Two factor ANOVA, n.s., n=6 per group.