Arginine-vasopressin expressing neurons in the murine suprachiasmatic nucleus exhibit a circadian rhythm in network coherence in vivo

The suprachiasmatic nucleus (SCN) is composed of functionally distinct subpopulations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize in vivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in arginine vasopressin (AVP)-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using in vivo electrical recordings, likely reflect burst firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among ne..., Image Analysis and Statistics
Image Correction, ROI Definition and Production of Intensity Traces
Raw image stacks acquired at 6.67 Hz were motion-corrected using Inscopix Data Processing software and five-minute recordings of images were exported as TIFF stacks. The TIFF stacks were imported into Igor Pro 8 (Wavemetrics Inc, Lake Oswego, Oregon) for further analysis. For each TIFF stack, a maximum projection image was then produced by finding the maximum intensity for each pixel during the 5-minute recording. This maximum projection image was used to identify regions of interest (ROIs) and regions of background. For a particular animal, ROI locations could vary slightly over the 24–48 hours of experimentation, and thus, ROI locations could be slightly corrected from 3-hour time point to time point. Background was subtracted from each image in the stack by doing a two-dimensional interpolation of background intensities based on the background regions outlined in the maximum projection ..., θIgor is needed to access .pxp data files and run analysis code as provided. We also provide an open-source HDF5 (https://en.wikipedia.org/wiki/Hierarchical_Data_Format) version of each .pxp file, each with the suffix .h5xp.  Â

视交叉上核（suprachiasmatic nucleus, SCN）由功能迥异的γ-氨基丁酸能神经元（GABAergic neurons）亚群构成，这些神经元形成了负责同步哺乳动物绝大多数生理与行为昼夜节律的神经网络。截至目前，学界对于SCN的节律特性中哪些由单个SCN神经元产生、哪些源于网络内神经元相互作用的认知仍较为匮乏。本研究利用在体微型显微镜技术，对自由活动清醒小鼠完整SCN内表达精氨酸加压素（arginine vasopressin, AVP）的神经元中荧光GCaMP标记的钙动态变化进行了检测。我们发现，SCN中的AVP神经元呈现周期性缓慢钙波；结合在体电生理记录结果，我们证实该钙波大概率对应神经元的爆发式放电。此外，我们观察到AVP神经元存在显著的功能异质性：其单个神经元节律不稳定，且群体水平的节律性相对较弱。网络分析揭示了细胞行为相关性（或称相干性）在ne...，图像分析与统计学
图像校正、感兴趣区域（ROI）划定与强度轨迹生成
以6.67 Hz帧率采集的原始图像序列通过Inscopix数据处理软件进行运动校正，并导出时长5分钟的图像记录为TIFF图像堆栈。将该TIFF图像堆栈导入Igor Pro 8（Wavemetrics公司，美国俄勒冈州莱克奥斯韦戈）以进行后续分析。针对每个TIFF图像堆栈，通过计算5分钟记录过程中各像素的最大强度值，生成最大投影图像。利用该最大投影图像确定感兴趣区域（ROI）与背景区域。针对单只实验动物，在24~48小时的实验周期内，其ROI位置可能会发生轻微偏移，因此可逐3小时时间点对ROI位置进行微调校正。通过基于最大投影图像中勾勒出的背景区域进行背景强度的二维插值，对图像堆栈中的每帧图像完成背景扣除……
需使用Igor软件以读取.pxp格式数据文件并运行所提供的分析代码。我们还为每个.pxp文件提供了开源的分层数据格式5（HDF5，https://en.wikipedia.org/wiki/Hierarchical_Data_Format）版本，其文件后缀为.h5xp。