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Abstract

With a growing and ageing population, neurodegenerative diseases like Alzheimer’s disease (AD) are going to appear more frequently in the next years. Currently approved drugs for the treatment of affected patients only mitigate the symptoms but do not prevent or cure the disease. Molecular mechanisms underlying the disease are still not completely understood, but there are indications that the immune system plays a key role in disease progression. Microglia, the resident immune cells of the brain, quite early in the disease progression prune away synaptic contacts. This loss of synaptic contacts is associated with a cognitive decline. Therefore, it is of great importance to study synaptic contacts since they could serve as an early indicator of the disease or as an efficacy readout in drug development. Here, we developed a robust and sensitive proximity ligation assay (PLA)-based method to quantify synaptic density on brain tissues of different types of mouse models. This required an optimized pair of antibodies recognizing pre- and postsynaptic markers which have to be in close vicinity to generate a PLA signal. We successfully identified marker pairs to visualize general as well as glutamatergic synapses. Important improvements of the PLA method included a comparison of different tissue preparation methods, buffers, probe coupling, mounting medium, heat distribution during hybridization, liquid barrier preparation, suitable controls, scanning and image analysis parameters. Upon development of a robust and reliable PLA protocol to determine synaptic density, we assessed synaptic contacts in healthy young and adult C57Bl6/J mice, spatiotemporal changes in large brain areas in two different AD models (Aβ and tau transgenic models), and finally changes in synaptic density after neurotoxic insult by trimethyltin (TMT). Our most important findings were i) a higher number of synaptic contacts in the entorhinal cortex in developing compared to adult brains, ii) a decreased number of synaptic contacts in close vicinity to Aβ plaques, iii) a decreased synaptic density in cortical layer I in 6 months-old tau-transgenic animals, and iv) a dose-dependent increase of synaptic contacts in the hippocampus upon TMT insult after 72 hours. These findings represent important basic data sets on the spatiotemporal distribution of synaptic contacts in healthy and diseased brains and especially provide novel data re- garding synapse density in the different cortical layers. Furthermore, spatiotemporal data on the synaptic density upon neurotoxic insult and the cortical layers in the tau transgenic AD model were used for a detailed power analysis to calculate the sample size of future treatment studies. This might help to avoid unnecessary high numbers of animals in preclinical drug development studies and to determine the most suitable timepoints for the measurement of synaptic contacts. Altogether, the present work established the PLA as a novel important tool to assess synaptic contacts in the frame of AD research. Major advantages are its high precision, robustness, reliability, the possibility to analyze large brain areas, that it is easy to handle and can be used in a more economical way as comparable methods to determine synaptic contacts. Spatiotemporal data on the synaptic density generated by the PLA in non-diseased and diseased brain represent an important data source for future preclinical studies on AD.