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TitleAuthorsAbstractMethods SummaryReferencesMethodsAnimals and surgical proceduresMultiphoton imagingImage processing and data analysisImmunohistochemistryMethods ReferencesFigure 1 Appearance of a novel plaque is a rapid process.Figure 2 Microglia recruitment follows plaque formation.Figure 3 Plaque formation has no immediate effect on neuritic curvature.LETTERSRapid appearance and local toxicity of amyloid-bplaques in a mouse model of Alzheimer’s diseaseMelanie Meyer-Luehmann1, Tara L. Spires-Jones1, Claudia Prada1, Monica Garcia-Alloza1, Alix de Calignon1,Anete Rozkalne1, Jessica Koenigsknecht-Talboo2, David M. Holtzman2, Brian J. Bacskai1& Bradley T. Hyman1Senile plaques accumulate over the course of decades in the brainsof patients with Alzheimer’s disease. A fundamental tenet of theamyloid hypothesis of Alzheimer’s disease is that the deposition ofamyloid-b precedes and induces the neuronal abnormalities thatunderlie dementia1. This idea has been challenged, however, by thesuggestion that alterations in axonal trafficking and morpho-logical abnormalities precede and lead to senile plaques2. The roleof microglia in accelerating or retarding these processes has beenuncertain. To investigate the temporal relation between plaqueformation and the changes in local neuritic architecture, we usedlongitudinal in vivo multiphoton microscopy to sequentiallyimage young APPswe/PS1d9xYFP (B6C3-YFP) transgenic mice3.Here we show that plaques form extraordinarily quickly, over 24 h.Within 1–2 days of a new plaque’s appearance, microglia areactivated and recruited to the site. Progressive neuritic changesensue, leading to increasingly dysmorphic neurites over the nextdays to weeks. These data establish plaques as a critical mediator ofneuritic pathology.To explore the formation of amyloid plaques and to determine theeffects of newly formed dense-cored plaques on the microarchitec-ture of the brain, we have developed a novel in vivo multiphotonimaging technique. This recognizes newly formed plaques and allowsmonitoring of their immediate vicinity thereafter to determine therate of their formation and the temporal sequence of pathophysio-logical events. We imaged young (5- to 6-month-old) B6C3-YFPmice, an age when plaques begin to appear4(Fig. 1). We usedthree-colour imaging to establish fiduciary markers for repeatedimaging: YFP positive neurons, dendrites and axons in the cortex,methoxy-XO4-labelled fibrillar amyloid-b deposits in the par-enchyma and on vessel walls, and a fluorescent angiogram withTexas red dextran to image blood vessels. A volume of cortex (laminaI–III) that initially did not contain plaques was re-imaged until repeatimaging detected a new plaque, establishing its ‘birthday’. To ensurethat the appearance of a new plaque did not simply reflect a greaterdepth of imaging or a slightly different imaging volume, we wentthrough each image stack and compared them with previous ses-sions. New plaques were accepted only if a uniquely identifiablefiduciary point, such as a blood vessel or a dendritic process, couldbe unambiguously noted in a deeper imaging plane.We postulated that we would occasionally observe the appearanceand growth of new plaques within an imaging volume if the timeinterval between imaging sessions was long enough. From one weeklyimaging session to the next, most of the sites remained unchanged(Supplementary Fig. 1a–c). However, we identified 14 new plaques:instances in which a plaque appeared in a second imaging session in avolume that had clearly been unoccupied in the first images one weekearlier (Fig. 1a–c).We examined the spatial relation between newly identified plaquesand blood vessels. Measurements of the distance between vessel walland the edge of a plaque confirmed that dense-core plaques developclose to but not within blood vessels (9.1 6 3.9 mm from blood ves-sels). As a control, 70 randomly placed, plaque-sized objects had anaverage distance of 8.4 6 11.2 mm from a vessel. New plaques there-fore do not form any closer to vessels than would be expected bychance, in accord with an earlier study of human Alzheimer’s dis-ease5. Furthermore, multiphoton microscopic images showed thatnewly formed plaques were not penetrated by blood vessels6, suggest-ing only a limited direct role of blood vessels in the formation ofdense-core plaques.To examine whether the phenotype of plaque formation in as shorta period as one week was unique to the aggressive APP/PS1 trans-genic mouse model, we used a mouse line that has a slower progres-sion of disease (Tg2576)7. Seven Tg2576 transgenic mice (11 months)were imaged weekly, and fourteen additional new plaques wereobserved, suggesting that the rapid plaque formation is not restrictedto one mouse model (Supplementary Fig. 2).We next imaged the B6C3-YFP mice on a daily basis for up tosix days in a row and/or on a weekly basis for up to three weeks(Fig. 1d–i). To our surprise, senile plaque formation is a very rapidevent, with five new plaques appearing precipitously within 24 h afterthe last imaging session. However, plaque formation is a rare event.Combining our experiments of daily and weekly imaging, a total of 26new plaques were found in 14 animals over 238 sites, imaged a total of1,285 times.These newly formed plaques were then re-imaged over days toweeks to determine their growth pattern. Measurements of plaquearea over several imaging sessions revealed that they do not change insize after about the first 24 h, regardless of whether they had small orlarge diameters when they were first imaged (Fig. 1j). To examinewhether imaging procedures had affected the observed plaque char-acteristics, we compared in vivo meas ures with those obtained frompost-mortem analysis of mice either after our imaging protocols orwith naive mice that had never been imaged. These analyses con-firmed that the newly formed plaques are not significantly different inarea or diameter from those observed post mortem and thereforerepresent typical plaques (Supplementary Fig. 3 ). Furthermore, thesize distribution of new plaques compares well with that of all plaquesseen post mortem in non-imaged mice (Supplementary Fig. 3). Theshape of this histogram is also quite similar to that observed inanalyses of human patients with Alzheimer’s disease8.Several studies have shown that microglia surround senile plaquesboth in patients with Alzheimer’s disease and transgenic mouse mod-els9,10although their role remains


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