Signaling at A-kinase anchoring proteins organizes anesthesia-sensitive memory in Drosophila

Abstract

The ubiquitous cAMP-protein kinase A (PKA) signaling pathway exhibits complex temporal requirements during the time course of associative memory processing. This directly raises questions about the molecular mechanisms that provide signaling specificity to this pathway. Here, we use Drosophila olfactory conditioning to show that divergent cAMP signaling is mediated by functionally distinct pools of PKA. One particular pool is organized via the PKA regulatory type II subunit at the level of A-kinase anchoring proteins (AKAPs), a family of scaffolding proteins that provides focal points of spatiotemporal signal integration. This AKAP-bound pool of PKA is acting within neurons of the mushroom bodies to support a late phase of aversive memory. The requirement for AKAP-bound PKA signaling is limited to aversive memory, but dispensable during appetitive memory. This finding suggests the existence of additional mechanisms to support divergence within the cAMP-PKA signaling pathway during memory processing. Together, our results show that subcellular organization of signaling components plays a key role in memory processing.

Figure 1.

AKAP-bound PKA-RII supports a distinct phase of aversive olfactory memory. Anchoring PKA-RII to AKAP complexes can be interfered with by ectopic expression of the UAS-eCOPR2 peptide which constitutes the docking domain of PKA-RII and, thus, dislodges endogenous PKA-RII from AKAP complexes. A, Pan-neuronal expression of the competitor (elav-Gal4/UAS-eCOPR2) results in altered memory kinetics: although memory is acquired to a wild-type extent, an accelerated loss of memory becomes obvious between 60 and 120 min after training, followed by a stable plateau at about half the level of wild-type-like performance at 120–180 min (p > 0.05 for time-points up to 60 min; p < 0.01 at 120 and 180 min). B, AKAP-bound PKA-RII is required within the mushroom bodies (247-Gal4/UAS-eCOPR2) to support aversive memory processing. The behavioral defect is indistinguishable from pan-neuronal expression (p < 0.01 at 120 min; when comparing 247-Gal4/UAS-eCOPR2 to elav-Gal4/UAS-eCOPR2: p values >0.05 at all time points). All data represent means ± SEM; n = 6 for all groups, except for wild-type n = 9. *p < 0.05; **p < 0.01.

Figure 2.

AKAP-bound PKA-RII acutely supports a distinct form of anesthesia-sensitive memory. A, Acute induction of the competitor peptide by use of the conditional blocker GAL80^ts produces loss of 3 h memory performance equal to permanent expression (p < 0.05 when comparing permissive to restrictive conditions; p > 0.05 when comparing performance of 247-Gal4, tub-Gal80ts/UAS-eCOPR2 flies under permissive conditions to elav-Gal4/UAS-eCOPR2 from Fig. 1A). B, Three hour memory is composed of two separable memory phases, ASM and ARM. Cold amnestic treatment leads to a significant decline of memory performance in wild-type flies, revealing (ARM, gray bars) and ARM plus ASM (black bars). In 247-Gal4/UAS-eCOPR2 flies, ASM is completely abolished regardless of amnestic treatment (p > 0.05). All data represent means ± SEM; n = 6 for all groups, except for wild-type n = 8. *p < 0.05; **p < 0.01.

Figure 3.

Appetitive and aversive memories are dissociated on the level of AKAP-bound PKA-R II signaling. Sugar reward leads to cAMP-dependent formation of appetitive memory. A, AKAP-bound PKA-R II is not required for performance of appetitive memory, as revealed by wild-type-like performance of flies expressing the competitor in various neuropils. Neither pan-neuronal (elav-Gal4/UAS-eCOPR2), nor MB-specific (247-Gal4/UAS-eCOPR2) or projection neuron-specific expression (GH146-Gal4/UAS-eCOPR2) impaired memory performance after appetitive training for up to 24 h (p values > 0.05). B, When cold-amnestic treatment is applied 30 min before testing 3 h memory, wild-type flies show a significant decline in performance when comparing treated (gray bars) to nontreated controls (black bars; p < 0.05). Pan-neuronal expression of the competitor in elav-Gal4/UAS-eCOPR2 flies shows memory performance indistinguishable from wild-type controls (p values > 0.05). All data represent means ± SEM; n = 6 for all groups, except for wild-type n = 9. *p < 0.05; **p < 0.01.

Figure 4.

Model of cAMP signaling divergence in olfactory memory processing. Aversive and appetitive olfactory memories are processed within the mushroom bodies (gray box). In addition to dissociation on the level of catecholamine transmitters, these two forms of behavioral plasticity display a high degree of similarity: both memories require cAMP signaling within the mushroom bodies for proper processing, and the amnesiac mutation distinguishes between an early and a late memory phase, respectively. Here, we have shown that the late phase is dissociated on the molecular level by involvement of AKAP-PKA-RII complexes. A, In case of aversive memory, AKAP-bound PKA-R II signals are exclusively required to sustain later phases of memory performance. A different PKA pool is necessary for performance at early stages. B, Appetitive memory is independent of the AKAP anchored pool of PKA-RII and requires a yet unknown molecular mechanism.