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Amyloid precursor protein (APP) is the precursor to Aβ plaques. The cytoplasmic domain of APP mediates attachment of vesicles to molecular motors for axonal transport. In APP-KO mice, transport of Mn2+ is decreased. In old transgenic mice expressing mutated human (APPSwInd) linked to Familial Alzheimer’s Disease, with both expression of APPSwInd and plaques, the rate and destination of Mn2+ axonal transport is altered, as detected by time-lapse manganese-enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. To determine the relative contribution of expression of APPSwInd versus plaque on transport dynamics, we developed a Tet-off system to decouple expression of APPSwInd from plaque, and then studied hippocampal to forebrain transport by MEMRI. Three groups of mice were compared to wild-type (WT): Mice with plaque and APPSwInd expression; mice with plaque but suppression of APPSwInd expression; and mice with APPSwInd suppressed from mating until 2 weeks before imaging with no plaque. MR images were captured before at successive time points after stereotactic injection of Mn2+ (3–5 nL) into CA3 of the hippocampus. Mice were returned to their home cage between imaging sessions so that transport would occur in the awake freely moving animal. Images of multiple mice from the three groups (suppressed or expressed) together with C57/B6J WT were aligned and processed with our automated computational pipeline, and voxel-wise statistical parametric mapping (SPM) performed. At the conclusion of MR imaging, brains were harvested for biochemistry or histopathology. Paired T-tests within-group between time points (p = 0.01 FDR corrected) support the impression that both plaque alone and APPSwInd expression alone alter transport rates and destination of Mn2+ accumulation. Expression of APPSwInd in the absence of plaque or detectable Aβ also resulted in transport defects as well as pathology of hippocampus and medial septum, suggesting two sources of pathology occur in familial Alzheimer’s disease, from toxic mutant protein as well as plaque. Alternatively mice with plaque without APPSwInd expression resemble the human condition of sporadic Alzheimer’s, and had better transport. Thus, these mice with APPSwInd expression suppressed after plaque formation will be most useful in preclinical trials.

Manganese-enhanced magnetic resonance imaging (MEMRI) holds exceptional promise for preclinical studies of brain-wide physiology in awake-behaving animals. The objectives of this review are to update the current information regarding MEMRI and to inform new investigators as to its potential. Mn(II) is a powerful contrast agent for two main reasons: (1) high signal intensity at low doses; and (2) biological interactions, such as projection tracing and neural activity mapping via entry into electrically active neurons in the living brain. High-spin Mn(II) reduces the relaxation time of water protons: at Mn(II) concentrations typically encountered in MEMRI, robust hyperintensity is obtained without adverse effects. By selectively entering neurons through voltagegated calcium channels, Mn(II) highlights active neurons. Safe doses may be repeated over weeks to allow for longitudinal imaging of brain-wide dynamics in the same individual across time. When delivered by stereotactic intracerebral injection, Mn(II) enters active neurons at the injection site and then travels inside axons for long distances, tracing neuronal projection anatomy. Rates of axonal transport within the brain were measured for the first time in "time-lapse" MEMRI. When delivered systemically, Mn(II) enters active neurons throughout the brain via voltage-sensitive calcium channels and clears slowly. Thus behavior can be monitored during Mn(II) uptake and hyperintense signals due to Mn(II) uptake captured retrospectively, allowing pairing of behavior with neural activity maps for the first time. Here we review critical information gained from MEMRI projection mapping about human

ELB) 26 27Key words: (at least 5) 28Magnetic Resonance Imaging, neuroimaging, statistical parametric mapping, serotonin transporter 29 knock-out mouse, predator stress, innate unconditioned fear, anxiety, mouse models 30 31Running title: Evolution of neural activity from fear to anxiety 32 33 34 2 Abstract 35 Life threatening fear after a single exposure evolves in a subset of vulnerable individuals to anxiety, which 36 may persist for their lifetime. Yet neither the whole brain's response to innate acute fear nor how brain 37 activity evolves over time is known. Sustained neuronal activity may be a factor in the development of 38 anxiety. We couple two experimental protocols to obtain a fear response leading to anxiety. Predator stress 39 (PS) is a naturalistic approach that induces fear in rodents; and the serotonin transporter knockout (SERT-40 KO) mouse responds to PS with sustained defensive behavior. Behavior was monitored before, during and at 41 short and long times after PS in WT and SERT-KO mice. Both genotypes responded to PS with defensive 42 behavior, and SERT-KO retained defensive behavior for 23 days, while wild type (WT) mice return to 43 baseline exploratory behavior by 9 days. Thus, differences in neural activity between WT and SERT-KO at 9 44 days after PS will identify neural correlates of persistent defensive behavior. We used longitudinal 45 manganese-enhanced magnetic resonance imaging (MEMRI) to identify brain-wide neural activity between 46 behavioral sessions. Mn 2+ accumulation in active neurons occurs in awake behaving mice and is 47 retrospectively imaged. To confirm expected effects of PS, behavior was monitored throughout. Following 48 the same two cohorts of mice, WT and SERT-KO, longitudinally allowed unbiased quantitative comparisons 49 of brain-wide activity by statistical parametric mapping (SPM). During natural behavior in WT, only low 50 levels of activity-induced Mn 2+accumulation were detected, while much more accumulation appeared 51 immediately after PS in both WT and SERT-KO, and evolved at 9 days to a new activity pattern at p<0.0001, 52 uncorr., T=5.4. Patterns of accumulation differed between genotypes, with more regions of the brain and 53 larger volumes within regions involved in SERT-KO than WT. A new computational segmentation analysis, 54 using our InVivo Atlas based on a manganese-enhanced MR image of a living mouse, revealed dynamic 55 changes in the volume of significantly enhanced voxels within each segment that differed between genotypes 56 across 45 of 87 segmented regions. At Day 9 after PS, the striatum and ventral pallidum were active in both 57 genotypes but more so in the anxious SERT-KO. SERT-KO also displayed sustained or increased volume of 58 Mn 2+ accumulation between Post-Fear and Day 9 in eight segments where activity was decreased or silenced 59 in WT. Staining of the same mice fixed at the conclusion of imaging sessions for c-fos, a marker of neural 60 activity, confirmed that MEMRI detected active neurons. Intensity measurements in 12 regions...

for technical support, and Neurosciences Associates for their serial sectioning and histologic staining of our mouse brains. We are grateful to the Beckman Institute at Caltech for support of the 11.7 Bruker MR scanner, to the Biological Imaging Center at Caltech, to Harry Gray for helpful discussions, and to Ralph Adolphs, Marianne Bronner and the Division of Biology and Biological Engineering at Caltech for supporting Bearer as a Visiting Associate.