CLOCK-dependent pathway in a single pair of LNd neurons instruct circadian-independent interval timing behavior.

Interval timing is a cognitive ability essential for behaviors such as mating, foraging, and decision-making, and it is distinct from circadian rhythm regulation. Despite the involvement of circadian clock genes in both interval timing and circadian rhythms, the mechanisms differentiating these functions remain unclear. Using Drosophila as a model, we demonstrate that the CLK/CYC heterodimer, but not PER/TIM, is essential for interval timing. Neuronal CLK/CYC expression is necessary and sufficient for sexual experience-dependent shorter mating duration (SMD) behavior. We identified that CLK/CYC expression in a single pair of ITP-positive LNd neurons is pivotal for SMD. These neurons are glutamatergic with output circuits to central brain regions. CLK variants lacking DNA binding motifs dissociate circadian rhythms from interval timing and sleep behaviors in these neurons. Our study uncovers a specialized circuit for interval timing and highlights the non-circadian functions of circadian clock genes. Statement of SignificanceThis study in Drosophila demonstrates that the CLK/CYC heterodimer is crucial for interval timing, distinct from circadian rhythm regulation. The research pinpoints a specific pair of neurons that are critical for shortened mating duration behavior, and it suggests that CLK variants can influence interval timing dissociation from circadian rhythm and sleep behaviors. HighlightsO_LICLK/CYC heterodimer are essential for interval timing. C_LIO_LISpecialized mechanism of interval timing behavior revealed. C_LIO_LIIdentified a pair of glutamatergic ITP-LNd neurons that independently regulate both interval timing and sleep. C_LIO_LIRevealed a novel mechanism by which CLK variants regulate sleep and interval timing. C_LI Graphical AbstractDifferent CLK/CYC mediate separate regulatory mechanism between circadian rhythm, sleep and interval timing in Drosophila melanogaster.