Evolution of brain-wide activity in the awake behaving mouse after acute fear by longitudinal manganese-enhanced MRI

Abstract

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 brains 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.