Defining Clock Neurons Within Distributed Circadian Circuits Through Multiscale Technologies.

The mammalian circadian system has traditionally been viewed as a hierarchical network organized around a master pacemaker in the suprachiasmatic nucleus (SCN), which synchronizes peripheral clocks to coordinate daily rhythms in physiology and behavior. While this framework has provided a foundational model for circadian regulation, recent advances in molecular profiling, circuit tracing, and neuronal manipulation reveal a more complex architecture. Multiple brain regions outside the SCN contain neurons with intrinsic circadian properties that actively participate in processing temporal information, challenging the classical central-peripheral model. These findings raise a fundamental conceptual question: what defines a circadian clock neuron? Here, we synthesize recent molecular, cellular, and systems-level studies to examine the defining features of neurons embedded within brain circadian clocks. By integrating high-resolution molecular profiling with functional circuit analysis, we propose an operational framework for identifying clock neurons across brain circuits. Specifically, we outline four criteria that characterize circadian clock neurons: molecular oscillation, autonomy, physiological rhythmicity, and circuit influence. This framework reflects current experimental capabilities and highlights how multiscale approaches-from single-cell transcriptomics to causal circuit manipulation-are reshaping our understanding of circadian organization in the brain. Clarifying the identity of clock neurons will be essential for mapping distributed circadian circuits and for developing targeted interventions for neurological and neurodegenerative disorders associated with circadian and sleep disruption.