Biological clock disruptions sound Alzheimer's alarm uva
. Mice were individually housed in cages containing running wheels in light-tight boxes which were illuminated with timed fluorescent lights . Wheel running data were collected and analyzed with ClockLab software . Activity onset was automatically detected by ClockLab software and when necessary, corrected by eye by an experimenter blinded to genotype and treatment group.
In jet lag re-entrainment trials, the onset of the dark phase was abruptly advanced by 6 h and running wheel activity was recorded for at least 7 days after light cycle shift. PSvalues were calculated using Prism software by fitting a sigmoid dose-response curve to onset times in days 0–6 after light cycle shift . Total running distance and preference for running in the dark were measured after all mice had completely re-entrained after a phase shift and were averaged across 2 days.
In masking trials, light intensity was decreased by wrapping fluorescent lights in neutral density filter films and measured with a spectrometer . Illuminance was measured in lux, and photon flux, summed from 380 to 780 nm, was calculated using formulas in the supplementary materials in. A 1hr light pulse was delivered from ZT13-14. Percent running in light was calculated by comparing to the activity measured during the same period of constant darkness on the preceding day for each mouse.
Primary antibodies used were anti-phospho-tau AT180 , anti-phospho-tau pThr231 , anti-RBPMS , anti-Iba1 , anti-amyloid D54d2 , and anti-melanopsin . Secondary antibodies used were donkey anti-rabbit AlexaFluor 594 , donkey anti-rabbit AlexaFluor 647 , donkey anti-mouse AlexaFluor 594 , and donkey anti-mouse AlexaFluor 647 .
Images were acquired with a Keyence BZ-X800 fluorescence microscope and images were stitched using BZ-X800 Analyzer software. Cell quantification was performed with ImageJ. For quantification of microglia, a 0.36 mmsquare was drawn in the ventromedial hypothalamus and all Iba1+ cells were counted. For quantification of RGCs in sectioned retinal tissue, 500–700 μm lines were drawn along the RGC layer beginning 300 μm to either side of the optic nerve exit point and all RBPMS+ cells were counted.
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