The accumulation of amyloid-␤ protein into plaques is a characteristic feature of Alzheimer's disease. However, the contribution of amyloid-␤ plaques to neuronal dysfunction is unknown. We compared intracellular recordings from neocortical pyramidal neurons in vivo in APP-Sw (Tg2576 transgenic mice overexpressing amyloid precursor protein with the Swedish mutation) transgenic mice to age-matched nontransgenic cohortsatageseitherbeforeorafterdepositionofcorticalplaques.Weshowthattheevokedsynapticresponseofneuronstotranscallosalstimuli is severely impaired in cortex containing substantial plaque accumulation, with an average 2.5-fold greater rate of response failure and twofold reduction in response precision compared with age-matched nontransgenic controls. This effect correlated with the presence of amyloid-␤ plaques and alterations in neuronal process geometry. Responses of neurons in younger APP-Sw animals, before plaque accumulation, were similar to those in nontransgenic controls. In all cases, spontaneous membrane potential dynamics were similar, suggesting that overall levels of synaptic innervation were not affected by plaques. Our results show that plaques disrupt the synchrony of convergent inputs, reducing the ability of neurons to successfully integrate and propagate information.

The lack of a specific biomarker makes preclinical diagnosis of Alzheimer's disease (AD) impossible, and it precludes assessment of therapies aimed at preventing or reversing the course of the disease. The development of a tool that enables direct, quantitative detection of the amyloid-␤ deposits found in the disease would provide an excellent biomarker. This article demonstrates the real-time biodistribution kinetics of an imaging agent in transgenic mouse models of AD. Using multiphoton microscopy, Pittsburgh compound B (PIB) was imaged with sub-m resolution in the brains of living transgenic mice during peripheral administration. PIB entered the brain quickly and labeled amyloid deposits within minutes. The nonspecific binding was cleared rapidly, whereas specific labeling was prolonged. WT mice showed rapid brain entry and clearance of PIB without any binding. These results demonstrate that the compound PIB has the properties required for a good amyloid-imaging agent in humans with or at risk for AD.positron emission tomography ͉ senile plaques ͉ Alzheimer's disease T he development of radioligands for in vivo imaging in the brain demands the demonstration of specificity for the target with good brain entry and exit kinetics. A technique that would allow direct assessment of the biodistribution kinetics with ligand-receptor specificity simultaneously would hasten development of new radioligands and confirm the utility of existing agents. Multiphoton microscopy is an in vivo imaging technique with very high spatial and temporal resolution that allows the precise determination of ligand binding specificity with sub-m resolution that can be used to characterize imaging probes in small animal models. In the context of Alzheimer's disease (AD), the ''receptors'' are amyloid-␤ plaques and neurofibrillary tangles (1, 2). AD is only diagnosed with certainty after death. The definitive diagnosis of this disease in living patients will depend on the detection of one or both of these neuropathological lesions either directly, by means of brain imaging, or indirectly, through a suitable biomarker that parallels their development (3). Progress with therapeutics aimed at removing these lesions will also be accelerated with the use of an end point to evaluate their efficacy (4). Significant progress has been achieved in developing imaging agents that enter the brain and target plaques and tangles specifically to allow direct imaging in humans (5-9). One promising compound, 2-(4Ј-methylaminophenyl)-6-hydroxy-benzothiazole (referred to as Pittsburgh compound B or PIB), a derivative of thioflavin T, labels plaques and cerebral amyloid angiopathy (CAA) in tissue sections from AD patients (6, 10, 11). The compound also crosses the blood-brain barrier (BBB), allowing peripheral administration of the amyloid-targeting reagent. This compound is readily labeled with carbon-11 for positron emission tomography (PET) scanning (10). Because the half-life of carbon-11 is Ϸ20 min, successful imaging will depend on very rapid brain entry,...

The accumulation of amyloid-beta into insoluble plaques is a characteristic feature of Alzheimer's disease. Neuronal morphology is distorted by plaques: rather than being essentially straight, they are substantially more curved than those in control tissue, their trajectories become altered, and they are frequently distended or swollen, presumably affecting synaptic transmission. Clearance of plaques by administration of antibodies to amyloid-beta is a promising therapeutic approach to the treatment of Alzheimer's disease, leading to stabilization of dementia by an unknown cellular mechanism. The effect of plaque clearance on plaque-induced neuronal alterations has not been studied previously. Here we show that both plaques and neuritic lesions are reversible in a strikingly short period of time after administration of a single dose of amyloid-beta antibody. Amyloid clearance and recovery of normal neuronal geometries were observed as early as 4 d and lasted at least 32 d after a single treatment. These results demonstrate that, once plaques are cleared, neuronal morphology is self-correcting and that passive antibody treatment has the potential to reverse neuronal damage caused by Alzheimer's disease and, hence, directly impact cognitive decline. Moreover, the rapid normalization of neuritic dystrophy suggests an unexpected degree of plasticity in the adult nervous system.

We describe the implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein-protein interactions on a spatial scale less than 10 nm using commercially available components.