Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.
Zilkha Neurogenetics Institute, University of Southern California, Los Angeles, CA, USA; California Institute of Technology, Pasadena, CA, USA.
Neuroimage. 2020 Nov 15;222:116975. doi: 10.1016/j.neuroimage.2020.116975. Epub 2020 May 28.
Life threatening fear after a single exposure evolves in a subset of vulnerable individuals to anxiety, which may persist for their lifetime. Yet neither the whole brain's response to innate acute fear nor how brain activity evolves over time is known. Sustained neuronal activity may be a factor in the development of a persistent fear response. We couple two experimental protocols to provoke acute fear leading to prolonged fear: Predator stress (PS), a naturalistic approach to induce fear in rodents; and Serotonin transporter knockout mouse (SERT-KO) that responds to PS with sustained defensive behavior. Behavior was monitored before, during and at short and long times after PS in wild type (WT) and SERT-KO mice. Both genotypes responded to PS with defensive behavior. SERT-KO retained defensive behavior for 23 days, while WT mice returned to baseline exploratory behavior by 9 days. Thus, differences in neural activity between WT and SERT-KO 9 days after PS identifies neural correlates of persistent defensive behavior, in mice. We used longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) to identify brain-wide neural activity associated with different behaviors. Mn accumulation in active neurons occurs in awake, behaving mice and is retrospectively imaged. Following the same two cohorts of mice, WT and SERT-KO, longitudinally allowed unbiased quantitative comparisons of brain-wide activity by statistical parametric mapping (SPM). During natural behavior in WT, only low levels of activity-induced Mn-accumulation were detected, while much more accumulation appeared immediately after PS in both WT and SERT-KO, and evolved at 9 days to a new activity pattern (p < 0.0001, uncorr., T = 5.4). Patterns of accumulation differed between genotypes, with more regions of the brain and larger volumes within regions involved in SERT-KO than WT. A new computational segmentation analysis, using our InVivo Atlas based on a manganese-enhanced MR image of a living mouse, revealed dynamic changes in the volume of significantly enhanced voxels within each segment that differed between genotypes across 45 of 87 segmented regions. At Day 9 after PS, the striatum and ventral pallidum were active in both genotypes but more so in the SERT-KO. SERT-KO also displayed sustained or increased volume of Mn accumulations between Post-Fear and Day 9 in eight segments where activity was decreased or silenced in WT. C-fos staining, an alternative neural activity marker, of brains from the same mice fixed at conclusion of imaging sessions confirmed that MEMRI detected active neurons. Intensity measurements in 12 regions of interest (ROIs) supported the SPM results. Between group comparisons by SPM and of ROI measurements identified specific regions differing between time points and genotypes. We report brain-wide activity in response to a single exposure of acute fear, and, for the first time, its evolution to new activity patterns over time in individuals vulnerable to persistent fear. Our results show multiple regions with dynamic changes in neural activity and that the balance of activity between segments is disordered in the SERT-KO. Thus, longitudinal MEMRI represents a powerful approach to discover how brain-wide activity evolves from the natural state either after an experience or during a disease process.
单次暴露后危及生命的恐惧会在一部分易感性个体中发展为焦虑，这种焦虑可能会持续一生。然而，人们既不知道大脑对先天急性恐惧的整体反应，也不知道大脑活动随时间的演变。持续的神经元活动可能是持久恐惧反应发展的一个因素。我们结合了两种实验方案来引发急性恐惧，导致持久恐惧：捕食者压力（PS），一种诱导啮齿动物恐惧的自然方法；和 5-羟色胺转运体敲除鼠（SERT-KO），对 PS 有持续的防御行为反应。在野生型（WT）和 SERT-KO 小鼠的 PS 前、PS 期间和 PS 后短时间和长时间监测行为。两种基因型对 PS 均表现出防御行为。SERT-KO 在 23 天内保留防御行为，而 WT 小鼠在 9 天内恢复基线探索行为。因此，PS 后 9 天 WT 和 SERT-KO 之间的神经活动差异确定了小鼠中持久防御行为的神经相关性。我们使用纵向锰增强磁共振成像（MEMRI）来识别与不同行为相关的全脑神经活动。在清醒、行为活跃的小鼠中，活性神经元中的锰积累发生，并且可以进行回顾性成像。对相同的两组（WT 和 SERT-KO）小鼠进行纵向研究，允许通过统计参数映射（SPM）对全脑活动进行无偏的定量比较。在 WT 的自然行为中，仅检测到低水平的活性诱导的 Mn 积累，而在 WT 和 SERT-KO 中，PS 后立即出现更多的积累，并且在 9 天时演变为新的活动模式（p<0.0001，未校正，T=5.4）。基因型之间的积累模式不同，WT 中涉及的大脑区域和区域内的体积更大。使用基于活体小鼠的锰增强磁共振图像的我们的 InVivo 图谱的新计算分割分析，揭示了在 PS 后 45 个分割区域中的 87 个中，每个区域内显著增强体素的体积发生了动态变化，这些变化在基因型之间存在差异。在 PS 后 9 天，纹状体和腹侧苍白球在两种基因型中均活跃，但在 SERT-KO 中更为活跃。SERT-KO 还在恐惧后和第 9 天之间的 8 个区域中显示出 Mn 积累的持续或增加体积，在 WT 中这些区域的活动减少或沉默。来自同一小鼠的大脑的 c-fos 染色，一种替代的神经活动标志物，固定在成像会话结束时，证实了 MEMRI 检测到活跃的神经元。12 个感兴趣区域（ROI）的强度测量结果支持 SPM 结果。SPM 的组间比较和 ROI 测量结果确定了特定的时间点和基因型之间的差异。我们报告了对单次急性恐惧暴露的全脑活动，并且首次报告了在易感性个体中，随着时间的推移，其向新的活动模式的演变。我们的结果显示了多个具有动态神经活动变化的区域，并且 SERT-KO 中节段之间的活动平衡紊乱。因此，纵向 MEMRI 代表了一种强大的方法，可以发现大脑在经历某种体验或疾病过程后如何从自然状态演变而来。
