The synchrony, robustness and precision generated by network interactions are necessary to sustain the role of the SCN as principal pacemaker. This coupling between cells generates emergent network-level properties of robust and precise oscillation (Hastings et al, 2018), which are adjusted to external time via direct retinal innervation (Abrahamson & Moore, 2001). These cellular TTFLs are coupled and synchronised across the SCN by paracrine and synaptic signals (Yamaguchi et al, 2003 Maywood et al, 2011 Noguchi et al, 2017 Patton et al, 2020). Individual cells maintain near 24 h autonomous rhythms through self-sustaining transcriptional/ translational feedback loops (TTFLs) (Partch et al, 2014), whereby Period (Per) and Cryptochrome (Cry) proteins negatively regulate their own expression, which is trans-activated by CLOCK:BMAL1 heterodimers. 20,000 circadian ( circa-diem) clock cells of the mammalian suprachiasmatic nucleus (SCN) (Abrahamson & Moore, 2001) co-ordinate sub-ordinate cellular clocks distributed across the body and thereby sustain adaptive daily rhythms of physiology and behaviour (Reppert & Weaver, 2002). These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination.

Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. This revealed their temporal plasticity, being up-regulated in circadian day. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood.

In the absence of network-level cellular interactions, the SCN fails as a clock. Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network.

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