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 anxiety. We couple two experimental protocols to obtain a fear response leading to anxiety. Predator stress (PS) is a naturalistic approach that induces fear in rodents; and the serotonin transporter knockout (SERT-KO) mouse responds to PS with sustained defensive behavior. Behavior was monitored before, during and at short and long times after PS in WT and SERT-KO mice. Both genotypes responded to PS with defensive behavior, and SERT-KO retained defensive behavior for 23 days, while wild type (WT) mice return to baseline exploratory behavior by 9 days. Thus, differences in neural activity between WT and SERT-KO at 9 days after PS will identify neural correlates of persistent defensive behavior. We used longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) to identify brain-wide neural activity between behavioral sessions. Mn2+ accumulation in active neurons occurs in awake behaving mice and is retrospectively imaged. To confirm expected effects of PS, behavior was monitored throughout. 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 Mn2+-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 at 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 anxious SERT-KO. SERT-KO also displayed sustained or increased volume of Mn2+ accumulation between Post-Fear and Day 9 in eight segments where activity was decreased or silenced in WT. Staining of the same mice fixed at the conclusion of imaging sessions for c-fos, a marker of neural activity, 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 Thus 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 anxiety. Our results demonstrate the power of longitudinal MEMRI to discover how brain-wide activity evolves during recovery or persistence of fear responses leading to anxiety.

中文翻译：

---

纵向锰增强MRI对急性恐惧后清醒行为小鼠的全脑活动演变

一次暴露后，威胁生命的恐惧会在一部分脆弱的个体中发展为焦虑，这种焦虑可能会持续一生。然而，既不知道整个大脑对先天性急性恐惧的反应，也不知道大脑活动随时间如何演变。持续的神经元活动可能是焦虑发展的一个因素。我们结合两个实验方案来获得导致焦虑的恐惧反应。捕食者应激（PS）是一种自然主义的方法，可引起啮齿动物恐惧。血清素转运蛋白敲除（SERT-KO）小鼠对PS的反应具有持续的防御行为。在WT和SERT-KO小鼠中在PS之前，期间以及之后和短期内监测行为。两种基因型均对PS采取防御行为，并且SERT-KO保留了23天的防御行为，而野生型（WT）小鼠则在9天后恢复了基线探索行为。因此，PS后9天时WT和SERT-KO之间神经活动的差异将确定持续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）来识别行为之间的全脑神经活动。活跃的神经元中Mn2 +的积累发生在清醒行为的小鼠中，并进行回顾性成像。为了确认PS的预期效果，始终对行为进行监控。在相同的两组小鼠中，WT和SERT-KO在纵向上通过统计参数映射（SPM）进行了全脑活动的无偏定量比较。在野生动物的自然行为期间，仅检测到低水平的活性诱导的Mn2 +积累，而在WT和SERT-KO中，PS后立即出现了更多的积累，并在9天时演化为p <0.0001的新活性模式，无相关性，T = 5.4。在基因型之间，积累的模式有所不同，与WT相比，SERT-KO涉及的大脑区域更多，区域内的体积更大。使用我们的InVivo Atlas基于活体小鼠的锰增强MR图像进行的新计算分段分析显示，每个分段内显着增强的体素体积的动态变化在87个分段区域中的45个基因型之间有所不同。PS后第9天，纹状体和腹侧苍白球在两种基因型中均活跃，而在焦虑的SERT-KO中则更为活跃。SERT-KO还显示了恐惧后和第9天之间八个部分中Mn2 +积累的持续或增加，其中WT中的活性降低或沉默。固定在c-fos（神经活动的标志物）成像会议结束时固定的同一只小鼠的染色证实MEMRI检测到了活跃的神经元。12个感兴趣区域（ROI）的强度测量结果支持了SPM结果。在通过SPM和ROI测量进行的组比较之间，确定了时间点和基因型之间存在差异的特定区域。因此，我们报告了一次暴露于急性恐惧中的全脑活动，并且首次报道了随着时间的推移其向新的活动模式的演变在易受焦虑困扰的人中。