FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons
The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3–4 and 8–9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner.
© 2015 Bosch, Martínez, Masachs, Teixeira, Fernaud, Ulloa, Pérez-Martínez, Lois, Comella, DeFelipe, Merchán-Pérez and Soriano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Received: 18 December 2014; Accepted: 29 April 2015; Published: 21 May 2015. We thank Tanya Yates for editorial assistance, Jorge G Peña for the EspINA software development and Alfonso Pérez-Escudero for critical discussion. This work was supported by grants from Spanish MINECO (BFU2008-03980 and SAF2013-42445R to ES, SAF2013-2010-19930 and PIE13-00027to JC, and BFU2012-34963 to JD), CIBERNED (to ES, JC, and JD), the Cajal Blue Brain Project, Spanish partner of the Blue Brain Project initiative from EPFL (to JD) and the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 604102 (Human Brain Project) (to JD). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
A sentence in the description of Figures 5A–D in the results section of Bosch et al. (2015) contained a minor error, which we hereby rectify (page 7, section "Three-Dimensional Analysis of Input Synapses onto Mature Adult-Generated Granule Cells", paragraph 3, lines 3–5). This modification does not alter any of the results or claims arisen in the original article, while adds coherence across the manuscript. It should read: "Spine and synapse sizes were distributed with a right-skewed curve, whereas sphericities distributed symmetrically around the means (Figures 5A–D)."