Presynaptic regulators in memory formation

Abstract

The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.

Figure 1.

Rab3 and Nrx adult knockdown in all KCs do not alter the gross architecture of AZs. (A) Quantification of Rab3 (F_(2,35) = 4.829, P = 0.0145, n = 12, post hoc Tukey's test Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Rab3, [*] P < 0.05) and Nrx (F_(2,35) = 6.353, P = 0.0046, n = 12, post hoc Tukey's test Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Rab3, [**] P < 0.01) staining intensity in MB lobes of flies presenting knockdown of either Rab3 or Nrx in adult MB lobes. Representative confocal images are shown with quantitative graphics. Scale bar, 50 µm. (B) Quantification of BRP^nc82 (F_(2,41) = 1.704, P = 0.1953, n = 14), Syd1 (F_(2,41) = 3.154, P = 0.0538, n = 14), and Unc13A (F_(2,41) = 0.5814, P = 0.5639, n = 14) staining intensity in MB lobes of Gal80^ts;OK107/RNAi-Rab3 flies and Gal80^ts;OK107/RNAi-Nrx flies compared to control flies. Representative confocal images are shown with quantitative graphics. Scale bar, 50 µm. Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad Software). Differences were tested by one-way ANOVA with Tukey's post hoc test. Asterisks denote the least significant of the pairwise post hoc comparisons between the genotype of interest and its controls following the usual nomenclature. P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01.

Figure 2.

Memory after postdevelopmental KCs knockdown of Rab3 and Nrx. (A) Flies expressing RNAi-Rab3 in the adult MB lobes present defect of STM (F_(2,23) = 6.701, P = 0.0056, n = 8, post hoc Tukey's multiple comparisons test, Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Rab3, [**] P < 0.01, Gal80^ts;OK107/RNAi-Rab3 vs. +/RNAi-Rab3, [*] P < 0.05), MTM 3 h (F_(2,38) = 12.32, P < 0.0001, n ≥ 11, post hoc Tukey's multiple comparisons test, Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Rab3, [***] P < 0.001, Gal80^ts;OK107/RNAi-Rab3 vs. +/RNAi-Rab3, [**] P < 0.01), and LTM (F_(2,17) = 6.895, P = 0.0075, n = 6, post hoc Tukey's multiple comparisons test, Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Rab3, [**] P < 0.01, Gal80^ts;OK107/RNAi-Rab3 vs. +/RNAi-Rab3, [*] P < 0.05) but normal MTM 1 h (F_(2,28) = 0.2027, P = 0.7406, n ≥ 9) after 5 d of induction. Without induction, those flies have normal STM (F_(2,19) = 0.1396, P = 0.8707, n ≥ 4), MTM 3 h (F_(2,23) = 0.7502, P = 0.4845, n = 8), and LTM (F_(2,17) = 0.6034, P = 0.5597, n = 6). Half-scores are presented in Supplemental Table S1. (B) Flies expressing RNAi-Rab3 in the adult αβ lobes present normal STM (F_(2,17) = 2.385, P = 0.1260, n = 6) and MTM 1 h (F_(2,15) = 0.3770, P = 0.6932, n ≥ 5), whereas MTM 3 h (F_(2,17) = 6.035, P = 0.0119, n = 6, post hoc Tukey's multiple comparisons test, Gal80^ts;c739/+ vs. Gal80^ts;c739/RNAi-Rab3, [*] P < 0.05, Gal80^ts;c739/RNAi-Rab3 vs. +/RNAi-Rab3, [*] P < 0.05) and LTM (F_(2,17) = 31.42, P < 0.0001, n = 6, post hoc Tukey's multiple comparisons test, Gal80^ts;c739/+ vs. Gal80^ts;c739/RNAi-Rab3, [***] P < 0.001, Gal80^ts;c739/RNAi-Rab3 vs. +/RNAi-Rab3, [***] P < 0.001) are decreased after 5 d of induction. Without induction, those flies have normal MTM 3 h (F_(2,17) = 2.615, P = 0.1061, n = 6) and LTM (F_(2,17) = 0.7554, P = 0.4869, n = 6). Half-scores are presented in Supplemental Table S2. (C) Flies expressing RNAi-Nrx in the adult MB lobes exhibit defect of STM (F_(2,17) = 5.671, P = 0.0146, n = 6, post hoc Tukey's multiple comparisons test, Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Nrx, [*] P < 0.05, Gal80^ts;OK107/RNAi-Nrx vs. +/RNAi-Nrx, [*] P < 0.05), MTM 1 h (F_(2,42) = 14.80, P < 0.0001, n ≥ 12, post hoc Tukey's multiple comparisons tests, Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Nrx, [***] P < 0.001, Gal80^ts;OK107/RNAi-Nrx vs. +/RNAi-Nrx, [***] P < 0.001), and 3 h (F_(2,26) = 6.363, P = 0.0061, n ≥ 8, post hoc Tukey's multiple comparisons test, Gal80^ts;OK107/+ vs. Gal80^ts;OK107/RNAi-Nrx, [*] P < 0.05, Gal80^ts;OK107/RNAi-Nrx vs. +/RNAi-Nrx, [*] P < 0.05). Those flies present normal LTM (F_(2,17) = 1.066, P = 0.3692, n = 6). After staying at 18°C, the STM (F_(2,30) = 0.8680, P = 0.4308, n ≥ 9), MTM 1 h (F_(2,26) = 0.5975, P = 0.5582, n ≥ 8), and 3 h (F_(2,23) = 0.5992, P = 0.5584, n = 8) of those flies appear normal. Half-scores are presented in Supplemental Table S3. Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad Software). Differences were tested by one-way ANOVA with Tukey's post hoc test. Asterisks denote the least significant of the pairwise post hoc comparisons between the genotype of interest and its controls following the usual nomenclature. P > 0.05, (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001. The gray squares indicate experiments in which noninduced controls were not needed.

Figure 3.

The TARGET system offers a reversal of the behavior after a return to the permissive temperature. (A) Gal80^ts;OK107/RNAi-Rab3 flies show normal STM 10 d at 18°C after 5 d of induction at 30°C (F_(2,14) = 2.972, P = 0.0894, n = 5). (B) Gal80^ts;OK107/RNAi-Nrx flies show normal STM 10 d at 18°C after 5 d of induction at 30°C (F_(2,14) = 1.577, P = 0.2466, n = 5). Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad Software). Differences were tested by one-way ANOVA with Tukey's post hoc test. Half scores are presented in Supplemental Table S4.