Inhibitory-modulatory coupling generates persistent activity during working memory

Abstract Working memory requires the stable maintenance of neural representations across temporal gaps, yet the circuit mechanisms that generate and stabilize persistent activity remain unsolved. Prevailing models emphasize recurrent excitation as the principal substrate of persistence, but how inhibitory and modulatory interactions shape the stability of temporal dynamics is unclear. Here, using trace conditioning in Drosophila, a working memory-dependent form of associative learning, we identify reciprocal inhibition as a circuit mechanism for sustaining persistent activity. In trace conditioning, a “trace” interval separates the conditioned and unconditioned stimuli, requiring maintenance of a neural representation across the trace interval, to support learning. Combining virtual-reality behavior, targeted neurogenetic perturbations, in vivo two-photon calcium imaging, and real-time neurotransmitter measurements, we uncover a reciprocal inhibitory microcircuit within the ellipsoid body that is selectively engaged during trace, but not delay (overlapping CS-US), conditioning. During the trace interval, ER2/4m neurons exhibit sustained activity, while reciprocally connected ER3/4d neurons show progressively strengthened suppression, forming a dynamically stabilized inhibitory loop. Disrupting GABA synthesis or reception within this circuit abolishes persistent activity and impairs trace learning, demonstrating the causal requirement for reciprocal inhibition in working memory maintenance. We further show that glutamatergic and nitric oxide signaling enhance inhibitory efficacy during the trace interval. In vivo neurotransmitter imaging reveals temporally structured dynamics in which glutamatergic signaling precedes and amplifies sustained GABAergic inhibition, consistent with modulatory stabilization of circuit persistence. Together, these findings identify reciprocal inhibition, reinforced by modulatory signaling, as a core circuit mechanism for dynamically stabilizing persistent neural representations. Our results challenge excitation-centric models of working memory and establish inhibitory-modulatory loops as a fundamental substrate for maintaining memory traces across time. Competing Interest Statement The authors have declared no competing interest.