Photonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo 060-8638, Japan.
Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21498-503. doi: 10.1073/pnas.1214415110. Epub 2012 Dec 4.
The circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) is a hierarchical multioscillator system in which neuronal networks play crucial roles in expressing coherent rhythms in physiology and behavior. However, our understanding of the neuronal network is still incomplete. Intracellular calcium mediates the input signals, such as phase-resetting stimuli, to the core molecular loop involving clock genes for circadian rhythm generation and the output signals from the loop to various cellular functions, including changes in neurotransmitter release. Using a unique large-scale calcium imaging method with genetically encoded calcium sensors, we visualized intracellular calcium from the entire surface of SCN slice in culture including the regions where autonomous clock gene expression was undetectable. We found circadian calcium rhythms at a single-cell level in the SCN, which were topologically specific with a larger amplitude and more delayed phase in the ventral region than the dorsal. The robustness of the rhythm was reduced but persisted even after blocking the neuronal firing with tetrodotoxin (TTX). Notably, TTX dissociated the circadian calcium rhythms between the dorsal and ventral SCN. In contrast, a blocker of gap junctions, carbenoxolone, had only a minor effect on the calcium rhythms at both the single-cell and network levels. These results reveal the topological specificity of the circadian calcium rhythm in the SCN and the presence of coupled regional pacemakers in the dorsal and ventral regions. Neuronal firings are not necessary for the persistence of the calcium rhythms but indispensable for the hierarchical organization of rhythmicity in the SCN.
下丘脑视交叉上核（SCN）中的昼夜节律起搏器是一个分层多振荡器系统，其中神经元网络在表达生理和行为的相干节律方面起着至关重要的作用。然而，我们对神经元网络的理解仍然不完整。细胞内钙介导输入信号，例如相位重置刺激，进入涉及时钟基因的核心分子环，以产生昼夜节律，以及从环路输出到各种细胞功能的信号，包括神经递质释放的变化。使用具有遗传编码钙传感器的独特大规模钙成像方法，我们可视化了培养物中整个 SCN 切片表面的细胞内钙，包括自主时钟基因表达无法检测到的区域。我们在 SCN 中在单细胞水平上发现了昼夜钙节律，其拓扑特异性较大，腹侧区域的振幅更大，相位延迟更大。尽管用河豚毒素（TTX）阻断神经元放电，但节律的稳健性降低但仍然存在。值得注意的是，TTX 使 SCN 背侧和腹侧之间的昼夜钙节律分离。相比之下，间隙连接阻滞剂 carbenoxolone 对单细胞和网络水平的钙节律仅有较小的影响。这些结果揭示了 SCN 中昼夜钙节律的拓扑特异性以及背侧和腹侧区域存在耦合的区域起搏器。神经元放电对于钙节律的持续存在不是必需的，但对于 SCN 中节律的分层组织是必不可少的。
