- Four pairs of DN1s are required by the 24-hour appetitive memory extinction
- Activation of these DN1s potentiates the extinction induced by a single training
- The downstream SIFa neurons relay the mediation of extinction from DN1s
- Multiple trials of extinction training evoke activity in one to three DN1s
Memory forms when a previously neutral stimulus (CS+) becomes competent to predict a biologically potent stimulus (US). But if the CS+ is repeatedly presented without the US after the memory formation, this memory will be suppressed by newly formed extinction memory. The striking feature of extinction learning is that it requires repeated trials to robustly form extinction. Extended repetition only yields memories that remain transient in nature, thus imposing challenges in understanding the underlying mechanisms of extinction learning. Here, we took advantage of the versatile genetic tools and the well characterised circadian system of Drosophila to link these unique features to clock neurons. We report that inhibiting the activity of clock neurons blocks the formation of extinction memory. Further investigation attributes this role to a subset of cryptochrome-positive dorsal neurons 1 (DN1s) and their downstream SIFamide neurons. The requirement of clock neurons comes from a gating mechanism of extinction for a single extinction learning trial robustly causes typical extinction when coupled with acute activation of DN1s, as marked by the initially enhanced, but eventually diminished memory suppression. Accordingly, we detected specific neural responses to extinction training in a few DN1s via calcium imaging fulfilled by the TRIC tool, but not in dorsal neurons 2 or dorsolateral neurons. Based on these findings, we propose that in extinction of appetitive long-term memory, multiple trials of extinction learning robustly activate DN1 clock neurons to open the gate of extinction, which may contribute to the transient nature of extinction memory.