Identifying spatial and temporal organization in the circadian clock (Commentary on Pauls et al.).
Identifying spatial and temporal organization in the circadian clock (Commentary on Pauls et al.).
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DOI：
10.1111/ejn.12670
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发表时间：
2014-08
期刊：
影响因子：
--
通讯作者：
Piggins HD
中科院分区：
文献类型：
--
作者：
Piggins HD
为了组织生理和行为中的昼夜节律（~ 24小时），视交叉上核（SCN）中的大脑主昼夜节律钟必须像熟练的守门员或雕塑家一样，在时间和空间上协调和连接其信号。单个SCN神经元包含细胞内的分子时钟，但这数千个细胞的自主振荡器需要细胞间的通信来同步它们的计时。SCN如何实现跨其解剖结构的细胞内和细胞间信号的整合还远未被理解。Pauls等人的当前研究（2014）提出了新的分析工具，询问SCN的时空组织。与许多脑结构不同，SCN在体外培养中维持其功能。事实上，从携带分子钟的生物发光报告基因的啮齿动物（如PER 2：：Luc小鼠）制备的SCN外植体在生物发光中维持约24小时的节律。虽然从这样的外植体的photoveomicroscopy记录是视觉上有吸引力的，他们提出了相当大的挑战，分析作为逐帧跟踪单个SCN细胞中的基因表达的周期是时间和劳动密集型。在这里，作者开发了一种快速，自动化的方法，似乎可以规避这些问题。为了做到这一点，作者从新生儿PER 2：：Luc SCN外植体中进行了摄影视频显微镜记录，并应用图像处理，然后基于发光信号的相似性进行聚类和光谱聚类分析，以生成整个SCN切片中PER 2表达的可量化图。然后，他们表明，他们的自动化程序产生的结果与之前对这些记录中细胞节律的手动评估一致。这是令人欣慰的，因为它表明这两种方法产生类似的结果。然而，自动化程序要快得多，并避免了SCN区域固有的采样不足，这通常发生在基于推定的单细胞的手动分析中。随后，他们确定在SCN内有一个明显的空间组织，使得SCN切片的近端区域可以被分解成类似生物发光信号的“簇”，这些信号在记录的几天内持续存在。有趣的是，当昼夜节律的时间尺度被过滤掉时，这种空间组织仍然存在。光谱嵌入分析表明，空间和时间组织之间有很强的关系。不出所料，在缺乏功能性分子钟的动物的SCN记录中，没有时间组织，但有趣的是，仍然检测到空间组织。在缺乏关键细胞间信号的小鼠SCN的一些记录中，检测到一些时间组织，而缺乏空间组织。总之，本研究报告了一种快速，有效和复杂的方法来分析SCN组织外植体中基因/蛋白质表达的空间和时间组织。这种方法揭示了空间和时间的影响之间的重要的相互关系，也显示了如何在细胞内或细胞间信号不存在时，两者是可分离的。一个警告是，目前还不清楚这种分析所揭示的空间组织是否识别了SCN的功能不同区域，或者仅仅反映了表达时钟基因的细胞的差异密度。毫无疑问，这种方法的后续迭代可以集成这些信息。
To organize circadian (~ 24 h) rhythms in physiology and behavior, the brain’s master circadian clock in the suprachiasmatic nuclei (SCN) must, like a proficient goalkeeper or sculptor, coordinate and connect its signals in both time and space. Individual SCN neurons contain the intracellular molecular clockworks, but these several thousand cell autonomous oscillators require intercellular communication to synchronize their timekeeping. How the SCN achieves the integration of intracellular and intercellular signals across its anatomical structure is far from understood. The current study by Pauls et al.(2014) presents new analytical tools to interrogate the spatiotemporal organization of the SCN. Unlike many brain structures, the SCN sustains its function ex vivo in culture. Indeed, SCN explants prepared from rodents bearing bioluminescent reporters of the molecular clock (such as PER2:: Luc mice) sustain~ 24 h rhythms in bioluminescence. Although photovideomicroscopy recordings from such explants are visually appealing, they present considerable challenges for analysis as frame-by-frame tracking of the cycles of gene expression in individual SCN cells is both time-and labour-intensive. Here, the authors develop a fast, automated approach that appears to circumvent such problems. To do this, the authors took photovideomicroscopy recordings made from neonatal PER2:: Luc SCN explants and applied image processing followed by a cluster and spectral cluster analysis, based on similarity of luminescence signals, to generate quantifiable maps of PER2 expression throughout entire SCN slices. They then show that their automated procedure generates results consistent with previous manual assessment of cellular rhythms in these recordings. This is reassuring as it indicates that both approaches yield similar outcomes. However, the automated procedure is much faster and avoids the inherent undersampling of SCN regions that often arises in manual analysis based on putative single cells. Subsequently they determined that there is a marked spatial organization within the SCN such that proximal regions of the SCN slice can be resolved into ‘clusters’ of similar bioluminescence signal that persist over several days of recording. Intriguingly, when circadian time scales were filtered out, this spatial organization remained. Spectral embedding analysis showed that there was a strong relationship between spatial and temporal organization. Unsurprisingly, in SCN recordings from animals lacking a functional molecular clock, there was no temporal organization, but interestingly, a spatial organization was still detected. In some recordings from the SCN of mice lacking a key intercellular signal, some temporal organization was detected, whereas spatial organization was lacking. In summary, the current study reports a quick, efficient and sophisticated methodology to analyse the spatial and temporal organization of gene/protein expression in SCN tissue explants. This approach reveals the important inter-relationship between spatial and temporal influences and also shows how the two are separable when intracellular or intercellular signals are absent. One caveat is that it is unclear whether the spatial organization uncovered by such analysis identifies functionally distinct areas of the SCN or simply reflects the differential density of cells expressing clock genes. No doubt subsequent iterations of this approach can integrate this information.
影响因子：
3.4
作者：
Pauls, S.;Foley, N. C.;Silver, R.
通讯作者：
Silver, R.