As with [4Cl-D-Phe6, Leu17] VIP, we first determined the efficacy and side effects of GABA antagonism within the context of our preparation. LD12:12 slices were cultured with

either vehicle (ddH20) or 200 μM of the GABAA receptor antagonist bicuculline (BIC), and then provided with vehicle (ddH20) or 20 μM GABA at the time of the fourth peak in vitro. GABA produced a phase delay in the PER2::LUC rhythm, consistent with previous results (Liu and Reppert, 2000), and this phase delay was blocked by BIC (Figure S6C). Consistent with previous research (Aton et al., 2006), BIC did not alter the rhythmic properties of SCN core cells or decrease the number of rhythmic cells LY2157299 manufacturer within LD12:12 slices (Figure S6D). Thus, BIC application effectively suppresses GABAA signaling over time in vitro without altering single-cell ABT-199 mouse oscillatory function. To test whether GABAA signaling

contributes to network resynchronization in vitro, LD12:12 and LD20:4 slices were cultured with 200 μM BIC added to the medium. BIC did not eliminate photoperiod-induced changes in SCN organization or function (Figures 6F and S6E), but it did inhibit network resynchronization over time in vitro (Figures 6C and S6F). In particular, BIC attenuated the phase advance portion of the coupling response curve by 71%, an effect similar to that produced by TTX and larger than that produced by VIP receptor antagonism (Figures 6C and 7). This reveals that GABAA signaling contributes to network coupling when SCN core cells are close to antiphase. In contrast, BIC did not attenuate phase delays like TTX or the VIP receptor antagonist, and did not destabilize the steady-state portion of the coupling response curve like the VIP receptor antagonist (Figures 6C and 7), indicating that non-GABAA signaling mechanisms facilitate synchrony when the network is in less polarized states. Lastly, the steady-state

portion of the coupling Etomidate response curve is stable when both BIC and the VIP receptor antagonist are applied (Figures 6D and 7), indicating that the destabilization produced during VIP antagonism is a response caused by GABAA signaling. Collectively, this pattern of results suggests that GABAA signaling promotes network synchrony in an antiphase state, but opposes network synchrony in a steady-state configuration. This state-dependent role for GABAA signaling may account for previous results indicating that GABA is sufficient to synchronize dissociated SCN neurons (Liu and Reppert, 2000), but its absence does not desynchronize the SCN network under steady-state conditions (Aton et al., 2006). Here, we developed a functional assay of SCN coupling that uniquely captures the dynamic process by which SCN neurons interact.