Number and precise localization of postsynaptic receptors are key determinants of synaptic function. At the mouse neuromuscular junction - one of the best studied synapses - acetylcholin receptor (AChR) density and distribution have been determined by multiple approaches such as electronmicroscopy (Fertuck and Salpeter 1976) and electrophysiology (Kuffler and Yoshikami 1975). This makes the preparation ideal to test the suitability of dSTORM (van de Linde et al., 2011) for quantifying synaptic receptor density, that was previously estimated to reach up to 23 000 AChR/µm² at the endplate. We present an approach of evaluating AChR density and distribution by a combination of electrophysiology and superresolution light microscopy with a spatial resolution of 20 nm.

We focus on endplates of neonatal mice due to a simpler receptor distribution compared to adult endplates. We take advantage of a set of well characterized neurotoxins that show distinct binding selectivity to the different binding sites of the receptor (Blount and Merlie, 1989; Sine et al., 1995). To validate our optical method we correlate electrophysiology and superresolution light microscopy in a cell line expressing embryonal AChRs. We measured ACh responses in BC3H-1 cells and subsequently blocked AChRs with bungarotoxin-Alexa 647. We then used dSTORM to image and count labeled AChRs and correlate these results to the electrophysiologically obtained data. Furthermore we determine AChR number by dSTORM at endplates of P5 C57Bl6 mice. Our data suggests that quantitative dSTORM is a suitable approach to estimate the number of receptors also at synapses, where estimates of receptor density are more challenging to obtain and assumed to change during synaptic plasticity.

References:

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Fertuck HC, Salpeter MM (1976) Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol 69:144-158.

Kuffler SW, Yoshikami D (1975) The distribution of acetylcholine sensitivity at the post-synaptic membrane of vertebrate skeletal twitch muscles: iontophoretic mapping in the micron range. J Physiol (Lond) 244:703-730.

Sine SM, Kreienkamp HJ, Bren N, Maeda R, Taylor P (1995) Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity. Neuron 15:205-211.

van de Linde S, Löschberger A, Klein T, Heidbreder M, Stevee W, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6:991-1009.