We then exploited this alternative

operational state to investigate the process of network resynchronization with real-time bioluminescence imaging of single SCN neurons over time in vitro. Our results reveal that SCN neurons interact in a dynamic manner through phase-dependent responses. Phase-dependent interactions are a staple of mathematical models of oscillator coupling, but it has been difficult to demonstrate experimentally, even in invertebrate pacemaker preparations. The finding that phase-dependent responses likewise characterize SCN responses to environmental cues Navitoclax supports the theory that this process is essential for all forms of synchronization (Hansel et al., 1995 and Smeal et al., 2010). Continued use of the coupling response curve developed here can provide further insight into the mechanisms by which SCN neurons influence one another to regulate network-level properties. By developing and employing this in vitro assay of SCN interactions, we have shown that

SCN neurons are coupled through nonredundant signaling mechanisms whose functional roles are influenced by the state of the network. Predating this work, there was strong evidence supporting a role for VIP in maintaining SCN network synchrony (Aton and Herzog, 2005). However, similar evidence for GABA was relatively modest, with no apparent role for GABAA signaling in maintaining network INK 128 research buy synchrony (Aton et al., 2006). Our results reveal that both VIP and GABAA signaling pathways contribute to SCN coupling, but that the roles of these SCN coupling factors are functionally distinct. Notably, we find that GABAA signaling contributes to SCN coupling specifically when the network is in a polarized state, but opposes synchrony under steady-state conditions. Further, our results indicate that VIP acts together with GABAA signaling to found promote resynchronization when the network is in an antiphase configuration, but opposes the actions of GABAA signaling

to promote network synchrony under steady-state conditions. The observation that signaling through VIP and GABAA pathways exerts opposing actions under steady-state conditions, with the latter destabilizing network synchrony, is consistent with a recent report investigating functional connections between SCN neurons in culture (Freeman et al., 2013). Our study complements and extends that work, demonstrating that GABAA signaling can either inhibit or promote network synchrony in a manner that depends on the state of the network. This state-dependent role of GABAA signaling may reflect phase-dependent resetting responses (i.e., GABA advances SCN cells near antiphase, but delays those in-phase), as predicted by the phase response curve for GABA (Liu and Reppert, 2000).