Drosophila Middle-Term Memory: Amnesiac is Required for PKA Activation in the Mushroom Bodies, a Function Modulated by Neprilysin 1

Abstract

In Drosophila, the mushroom bodies (MB) constitute the central brain structure for olfactory associative memory. As in mammals, the cAMP/PKA pathway plays a key role in memory formation. In the MB, Rutabaga (Rut) adenylate cyclase acts as a coincidence detector during associative conditioning to integrate calcium influx resulting from acetylcholine stimulation and G-protein activation resulting from dopaminergic stimulation. Amnesiac encodes a secreted neuropeptide required in the MB for two phases of aversive olfactory memory. Previous sequence analysis has revealed strong homology with the mammalian pituitary adenylate cyclase-activating peptide (PACAP). Here, we examined whether amnesiac is involved in cAMP/PKA dynamics in response to dopamine and acetylcholine co-stimulation in living flies. Experiments were conducted with both sexes, or with either sex. Our data show that amnesiac is necessary for the PKA activation process that results from coincidence detection in the MB. Since PACAP peptide is cleaved by the human membrane neprilysin hNEP, we searched for an interaction between Amnesiac and Neprilysin 1 (Nep1), a fly neprilysin involved in memory. We show that when Nep1 expression is acutely knocked down in adult MB, memory deficits displayed by amn hypomorphic mutants are rescued. Consistently, Nep1 inhibition also restores normal PKA activation in amn mutant flies. Taken together, the results suggest that Nep1 targets Amnesiac degradation to terminate its signaling function. Our work thus highlights a key role for Amnesiac in establishing within the MB the PKA dynamics that sustain middle-term memory (MTM) formation, a function modulated by Nep1.

SIGNIFICANCE STATEMENT The Drosophila amnesiac gene encodes a secreted neuropeptide whose expression is required for specific memory phases in the mushroom bodies (MB), the olfactory memory center. Here, we show that Amnesiac is required for PKA activation resulting from coincidence detection, a mechanism by which the MB integrate two spatially distinct stimuli to encode associative memory. Furthermore, our results uncover a functional relationship between Amnesiac and Neprilysin 1 (Nep1), a membrane peptidase involved in memory and expressed in the MB. These results suggest that Nep1 modulates Amnesiac levels. We propose that on conditioning, Amnesiac release from the MB allows, via an autocrine process, the sustaining of PKA activation-mediating memory, which subsequently is inactivated by Nep1 degradation.