A novel method for quantitative analysis of the molecular composition of individual, functionally characterised synapses
The biophysical properties of individual synapses vary tremendously, even if the synapses are made by the same pre- and postsynaptic cell types. To elucidate the molecular mechanisms underlying the functional heterogeneity, a high resolution, sensitive, diffusion-free, quantitative localization method needs to be developed that allows the determination of a large number of proteins in individual synapses. Array tomography, a method based on repeated immunofluorescent labelling of serial sections from acrylic resin-embedded tissue, permits the quantitative analysis of individual synapses at multiple rounds of labelling, but its sensitivity and applicability to functionally characterized synapses are limited. Here, we aimed to overcome the limitations of array tomography by developing a highly sensitive, multiplexed postembedding immunolocalization method. By searching the parameter space of different fixations, resin embedding, etching, retrieval and elution conditions, we demonstrate that etching the epoxy resin-embedded ultrathin sections with Na-ethanolate and treating them with SDS dramatically increased the labelling efficiency of synaptic proteins compared to acrylic resin-embedded tissues. The quantitative nature of the method was verified by confirming the known tight correlation between the spine volume and the amount of PSD95 in the postsynaptic density. We also show that this technique is ideally suited for the molecular characterization of synapses following paired recordings, 2-photon [Ca2+], or glutamate imaging. Here we describe the development of a highly sensitive, quantitative, diffusion-free localization method that allows the proteomic analysis of individual functionally characterized synapses.