, 1997, Lim et al., 1999 and Kasugai et al., 2010). The number of endogenous GABAAR complexes at synapses has been estimated to vary from 30 to as many as 200 (Nusser et al., 1997), and that of GlyRs from 10

to 70 (Singer and Berger, 1999 and Rigo et al., 2003). However, nothing is known about the absolute numbers of gephyrin molecules at inhibitory synapses or about the relative stoichiometry of receptors and scaffold proteins. Here, we make use of quantitative, dynamic, and three-dimensional (3D) nanoscopic imaging not only to determine the subsynaptic distribution of gephyrin and receptor complexes at inhibitory PSDs but also to count the number of gephyrin molecules 5-FU in vitro and receptor binding sites. With this project, our goal was to visualize inhibitory

synapses at superresolution and to extract detailed structural and quantitative information about the PSD. We carried out photoactivated localization microscopy (PALM) on rat dissociated spinal cord cultured neurons expressing photoconvertible constructs of the synaptic scaffold protein gephyrin (mEos2- or Dendra2-gephyrin). PALM was first conducted on fixed neurons as described in the Experimental Procedures section. The positions of single fluorophores were determined by Gaussian fitting of their point-spread function (PSF) and were corrected for lateral drifts using fiducial markers. The localization accuracy was estimated as the SD σ of multiple detections of the Selisistat mouse same fluorophore in subsequent image frames (Izeddin et al., 2011). The precision of localization heptaminol was marginally better for mEos2-gephyrin (σx = 11.2 ± 1.9 nm mean ± SD, σy = 11.9 ±

1.4 nm, n = 12 fluorophores) than for Dendra2-gephyrin (σx = 13.1 ± 2.1 nm, σy = 12.8 ± 2.0 nm, n = 11). When expressed in spinal cord neurons, mEos2-gephyrin and Dendra2-gephyrin accumulate in dense clusters that are visible by conventional fluorescence microscopy (Figure 1A). PALM imaging makes it possible to measure the sizes of these structures with high precision (spatial resolution, ∼25–30 nm). Image segmentation of the rendered PALM images indicates an apparent surface ranging from 0.01 to 0.1 μm2 (Figure 1B). The PALM experiments also revealed the presence of an additional population of gephyrin clusters below 0.01 μm2 that is not visible in the diffraction-limited images (Figures 1A and 1B). To determine the subcellular localization of both types of clusters, we combined PALM imaging with direct stochastic optical reconstruction microscopy (dSTORM) as described elsewhere (Izeddin et al., 2011). In these experiments, the presynaptic protein bassoon was labeled with Alexa 647-tagged antibodies. Dual-color PALM/STORM images show the apposition of the large gephyrin clusters with bassoon-positive structures, identifying them as inhibitory PSDs (Figure 1C).