Abstract
Circadian pacemaking in suprachiasmatic nucleus (SCN) neurons revolves around transcriptional/posttranslational feedback loops, driven by protein products of "clock" genes. These loops are synchronized and sustained by intercellular signaling, involving vasoactive intestinal peptide (VIP) via its VPAC2 receptor, which positively regulates cAMP synthesis. In turn, SCN cells communicate circadian time to the brain via a daily rhythm in electrophysiological activity. To investigate the mechanisms whereby VIP/VPAC2/cAMP signaling controls SCN molecular and electrical pacemaking, we combined bioluminescent imaging of circadian gene expression and whole-cell electrophysiology in organotypic SCN slices. As a potential direct target of cAMP, we focused on hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. Mutations of VIP-ergic signaling compromised the SCN molecular pacemaker, diminishing the amplitude and intercellular synchrony of circadian gene expression. These deficits were transiently reversed by elevation of cAMP. Similarly, cellular synchrony in electrical firing rates was lost in SCN slices lacking the VPAC2 receptor for VIP. Whole-cell current-clamp recordings in wild-type (WT) slices revealed voltage responses shaped by the conductance I(h), which is mediated by HCN channel activity. The influence of I(h) on voltage responses showed a modest peak in early circadian day, identifying HCN channels as a putative mediator of cAMP-dependent circadian effects on firing rate. I(h), however, was unaffected by loss of VIP-ergic signaling in VPAC2-null slices, and inhibition of cAMP synthesis had no discernible effect on I(h) but did suppress gene expression and SCN firing rates. Moreover, only sustained but not acute, pharmacological blockade of HCN channels reduced action potential (AP) firing. Thus, our evidence suggests that in the SCN, cAMP-mediated signaling is not a principal regulator of HCN channel function and that HCN is not a determinant of AP firing rate. VIP/cAMP-dependent signaling sustains the SCN molecular oscillator and action potential firing via mechanisms yet to be identified.