Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
J Biol Rhythms. 2011 Jun;26(3):210-20. doi: 10.1177/0748730411402810.
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.
视交叉上核（SCN）神经元的昼夜节律起搏围绕着转录/翻译后反馈环进行，由“时钟”基因的蛋白质产物驱动。这些环通过细胞间信号传递进行同步和维持，涉及血管活性肠肽（VIP）通过其 VPAC2 受体，该受体正向调节 cAMP 的合成。反过来，SCN 细胞通过电生理活动的日常节律向大脑传递昼夜时间。为了研究 VIP/VPAC2/cAMP 信号如何控制 SCN 分子和电起搏，我们在器官型 SCN 切片中结合了昼夜基因表达的生物发光成像和全细胞膜片钳电生理学。作为 cAMP 的潜在直接靶点，我们专注于超极化激活、环核苷酸门控（HCN）通道。VIP 能信号的突变削弱了 SCN 的分子起搏器，降低了昼夜基因表达的幅度和细胞间同步性。这些缺陷可被 cAMP 的升高短暂逆转。同样，缺乏 VIP 的 VPAC2 受体的 SCN 切片中，电发放率的细胞同步性丧失。在野生型（WT）切片中的全细胞膜片钳记录中，我们发现由 I(h)介导的电导引起的电压响应，I(h)由 HCN 通道活性介导。I(h)对电压响应的影响在早期昼夜节律日中表现出一个适度的峰值，表明 HCN 通道是 cAMP 对放电率的昼夜效应的潜在介导物。然而，在 VPAC2 缺失的切片中，I(h)不受 VIP 能信号丢失的影响，而 cAMP 合成的抑制对 I(h)没有明显影响，但确实抑制了基因表达和 SCN 放电率。此外，只有持续但不是急性的 HCN 通道阻断可降低动作电位（AP）放电。因此，我们的证据表明，在 SCN 中，cAMP 介导的信号传递不是 HCN 通道功能的主要调节剂，并且 HCN 不是 AP 放电率的决定因素。VIP/cAMP 依赖性信号通过尚未确定的机制维持 SCN 分子振荡器和动作电位放电。
