bioRxiv2025
Circadian clock genes are best known for regulating circadian rhythms, but they also play crucial roles in memory processes. This suggests that memory is modulated by neural networks containing clock neurons, although the underlying mechanisms remain unclear. In Drosophila melanogaster, approximately 240 clock neurons are grouped into at least eight distinct clusters. Among them, the dorsal-lateral neurons (LNds) are required for maintaining long-term memory (LTM). In contrast, the neuropeptide Pigment-dispersing factor (Pdf), expressed in both small and large ventral-lateral neurons (s-LNvs and l-LNvs, respectively), functions as a circadian output signal and is also essential for maintaining LTM. In addition, Pdf-expressing neurons (hereafter, Pdf neurons) release neurotransmitters other than Pdf, which are involved in LTM consolidation. However, the specific transmitters used by LNds and Pdf neurons in LTM processing have remained unknown. Here, we show that octopamine signaling from LNds is essential for LTM maintenance, whereas octopamine in Pdf neurons is essential for LTM consolidation. Temporally restricted knockdown of Tyramine {beta} hydroxylase (Tbh), the gene encoding the enzyme required for octopamine synthesis, disrupted LTM maintenance when targeted in LNds, whereas it impaired LTM consolidation when targeted in Pdf neurons. Notably, Tbh knockdown in LNds or Pdf neurons had minimal effects on circadian behavioral rhythms or sleep. These findings reveal that octopamine released from specific subtypes of clock neurons independently regulates distinct phases of LTM in Drosophila. Author SummaryAnimal memory formed through learning is stabilized by a process called consolidation and becomes long-term memory (LTM), which can persist for extended periods. The fruit fly Drosophila melanogaster is widely used in memory research, enabling the discovery of key genes and signaling pathways. In addition, Drosophila has contributed significantly to circadian rhythm research through studies of clock neurons and clock genes. Notably, clock neurons have been implicated in LTM regulation, suggesting a potential link between circadian clock neurons and memory function. However, the neural network connecting these processes remains poorly understood. Identifying the neurotransmitters released from clock neurons is critical to clarifying how these neurons affect memory function. In this study, we show that octopamine, the fly equivalent of the vertebrate neurotransmitter norepinephrine, released from two distinct groups of clock neurons regulates different phases of LTM in Drosophila: one group supports consolidation, whereas the other supports maintenance. Our findings demonstrate that clock neurons play an active role in memory regulation, and they provide new insights into the neural circuitry that controls LTM.