Temporally and Spatially Localized PKA Activity within Learning and Memory Circuitry Regulated by Network Feedback

Abstract

Dynamic functional connectivity within brain circuits requires coordination of intercellular signaling and intracellular signal transduction. Critical roles for cAMP-dependent protein kinase A (PKA) signaling are well established in the Drosophila mushroom body (MB) learning and memory circuitry, but local PKA activity within this well-mapped neuronal network is uncharacterized. Here, we use an in vivo PKA activity sensor (PKA-SPARK) to test spatiotemporal regulatory requirements in the MB axon lobes. We find immature animals have little detectable PKA activity, whereas postcritical period adults show high field-selective activation primarily in just 3/16 defined output regions. In addition to the age-dependent PKA activity in distinct α'/β' lobe nodes, females show sex-dependent elevation compared with males in these same restricted regions. Loss of neural cell body Fragile X mental retardation protein (FMRP) and Rugose [human Neurobeachin (NBEA)] suppresses localized PKA activity, whereas overexpression (OE) of MB lobe PKA-synergist Meng-Po (human SBK1) promotes PKA activity. Elevated Meng-Po subverts the PKA age-dependence, with elevated activity in immature animals, and spatial-restriction, with striking γ lobe activity. Testing circuit signaling requirements with temperature-sensitive shibire (human Dynamin) blockade, we find broadly expanded PKA activity within the MB lobes. Using transgenic tetanus toxin to block MB synaptic output, we find greatly heightened PKA activity in virtually all MB lobe fields, although the age-dependence is maintained. We conclude spatiotemporally restricted PKA activity signaling within this well-mapped learning/memory circuit is age-dependent and sex-dependent, driven by FMRP-Rugose pathway activation, temporally promoted by Meng-Po kinase function, and restricted by output neurotransmission providing network feedback.

Keywords: Drosophila; FMRP; Kenyon cell; Meng-Po; mushroom body; neurobeachin.

Figure 1.

Early life, sex-dependent PKA activity in Mushroom Body circuit regions. A, Schematic of MB lobes and defined MBON fields (dashed outlines) shown in three layers: α’/β’ (left), α/β (middle), and γ lobes (right). B, C, Representative images of MB lobes with OK107-Gal4 driving UAS-PKA-SPARK at 0 dpe (B) and 7 dpe (C). The MBON fields (dashed circles) and arrows delineate the α’1 and β’1 quantified regions. D, E, PKA-SPARK::GFP puncta number in both regions, including α’1 (D) and β’1 (E). Scatter plots show all data points and mean ± SEM. F, G, MB lobes with OK107-Gal4 driving UAS-PKA-SPARK in female (F) and male (G) at 7 dpe. H, I, Quantification of PKA-SPARK puncta in both α’1 (H) and β’1 (I). Sample size >15 fields in all conditions. Statistics show two-tailed t tests with Welch’s correction (H) or Mann–Whitney tests (D, E, I). Significance: ***p < 0.001.

Figure 2.

PKA-SPARK::GFP reporter levels constant across sex and age groups. A, Representative Western blotting comparing PKA-SPARK::GFP protein levels (anti-GFP) from 0 and 7 dpe time points, in both females and males with OK107-Gal4 driving UAS-PKA-SPARK::GFP. The protein loading control is α-Tubulin (α-tub). Probed proteins are indicated on the left and molecular weights on the right. B, PKA-SPARK::GFP protein levels normalized to the α-tub loading control. Scatter plots show all data points and mean ± SEM. Statistics show Brown–Forsythe and Welch ANOVA tests. Sample size: 9, all conditions. Significance: not significant (n.s.; p > 0.05).

Figure 3.

Localized MB lobe PKA activity signaling enabled by FMRP and Rugose. A, Representative images of MB lobes with OK107-Gal4 driving UAS-PKA-SPARK at 7 dpe in control (left) and dfmr1^50M null (right). Arrows point to α’1 and β’1 (Fig. 1A). PKA-SPARK::GFP puncta number in α’1 (B) and β’1 (C) regions. D, Similar confocal imaging comparison at 7 dpe in control (left) and rg^FDD null (right). E, F, Quantification of PKA-SPARK::GFP puncta number in α’1 (E) and β’1 (F) regions. Note that this comparison is done in males only owing to the X chromosome location of the rugose gene. Scatter plots show all data points and mean ± SEM. Sample size >12 fields in all conditions. Statistics show two-tailed t tests with Welch’s correction (B, C, F) or Mann–Whitney tests (E). Significance: ***p < 0.001.

Figure 4.

Localized circuit PKA activity increased with Meng-Po OE. A–F, Representative images of MB lobes with OK107-Gal4 driving UAS-PKA-SPARK at 0 and 7 dpe in control (A) and with targeted OE of UAS-dFMRP (B), UAS-hFMRP (C), UAS-ΔRGG-hFMRP (D), UAS-Rugose (Rg, E), and UAS-Meng-Po (MP, F). Arrows indicate the α’1 and β’1 regions (Fig. 1A) of normally heightened PKA activity. Arrowhead (F) points to the expanded PKA activity within the γ3 region. G, H, Quantification of PKA-SPARK::GFP puncta in α’1 (G) and β’1 (H) regions. Scatter plots show all data points and mean ± SEM. Sample size: >7 fields in every genotype and at every time point. Statistics show two-tailed t tests with Welch’s correction and Mann–Whitney tests (see statistical table; Table 1). Significance: ***p < 0.001.

Figure 5.

Localized MB lobe PKA activity signaling requires the Meng-Po kinase. A, Representative MB lobe images with OK107-Gal4 driving UAS-PKA-SPARK at both 0 dpe (left) and 7 dpe (right) in the genetic control background. Quantified MBON fields (dashed circles, left) and arrows indicate α’1 and β’1 regions. B, Representative MB lobes images with meng-po RNAi under the same conditions as in A. Scale bar: 10 μm. C, D, Quantification of PKA-SPARK::GFP puncta in α’1 (C) and β’1 (D) MBON fields. Scatter plots show all data points and mean ± SEM. Statistics show Kruskal–Wallis tests. Sample size: >9, all conditions. Significance: ***p < 0.001, not significant (n.s.; p > 0.05).

Figure 6.

Conditional shibire^ts neurotransmission block increases PKA activity. A–D, Representative images of MB lobes with OK107-Gal4 driving UAS-shibire^ts at the 0 and 7 dpe time points at the indicated adult rearing temperatures. Dotted outlines define additional MBON regions of elevated PKA activity detected with the PKA-SPARK reporter, and arrowheads indicate expanded α/α’ neuropils. E–H, Quantified number of PKA-SPARK:GFP puncta in defined MB lobe regions. Scatter plots show all data points and the mean ± SEM. Sample size >12 fields in every temperature condition. Statistics show Brown–Forsythe and Welch ANOVA tests (E, G) and Kruskal–Wallis tests (F, H). Significance: *p < 0.05, ***p < 0.001 and not significant (n.s.; p > 0.05).

Figure 7.

MB circuit PKA activity dramatically expanded by synaptic output block. A, B, MB lobes with OK107-Gal4 driving UAS-tetanus toxin light chain (TNT) at both 0 dpe (A) and 7 dpe (B). Arrows indicate expanded PKA activity regions detected with the PKA-SPARK reporter and dotted outlines indicate newly-recruited MBON regions. C–I, Quantification of PKA-SPARK::GFP puncta number in each defined MBON region, including α’1 (C), β’1 (D), γ1 (E), α’3 (F), α3 (G), β’2m (H), and β2 (I). Scatter plots show all data points and the mean ± SEM. Sample size >19 fields in every paired comparison. Statistics show Mann–Whitney tests. Significance: ***p < 0.001.