title: FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons.
abstract: 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.
authors.affiliation: Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain ; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain ; Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron (VHIR) Barcelona, Spain.
authors.forename: Carles
authors.initials: C
authors.lastname: Bosch
authors.affiliation: Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain.
authors.forename: Albert
authors.initials: A
authors.lastname: Martínez
authors.affiliation: Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain ; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain.
authors.forename: Nuria
authors.initials: N
authors.lastname: Masachs
authors.affiliation: Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain ; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain.
authors.forename: Cátia M
authors.initials: CM
authors.lastname: Teixeira
authors.affiliation: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain ; Laboratorio Cajal de Circuitos Corticales, Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, Campus de Montegancedo Madrid, Spain ; Instituto Cajal (Consejo Superior de Investigaciones Científicas) Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Fernaud
authors.affiliation: Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain ; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain.
authors.forename: Fausto
authors.initials: F
authors.lastname: Ulloa
authors.affiliation: Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain ; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Pérez-Martínez
authors.affiliation: Department of Neurobiology, University of Massachusetts Medical School Worcester, MA, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: Lois
authors.affiliation: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain ; Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron (VHIR) Barcelona, Spain ; Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona Bellaterra, Spain.
authors.forename: Joan X
authors.initials: JX
authors.lastname: Comella
authors.affiliation: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain ; Laboratorio Cajal de Circuitos Corticales, Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, Campus de Montegancedo Madrid, Spain ; Instituto Cajal (Consejo Superior de Investigaciones Científicas) Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: DeFelipe
authors.affiliation: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain ; Laboratorio Cajal de Circuitos Corticales, Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, Campus de Montegancedo Madrid, Spain ; Departamento de Arquitectura y Tecnología de Sistemas Informáticos, Escuela Técnica Superior de Ingenieros Informáticos, Universidad Politécnica de Madrid Madrid, Spain.
authors.forename: Angel
authors.initials: A
authors.lastname: Merchán-Pérez
authors.affiliation: Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain ; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Insituto de Salul Carlos III Madrid, Spain ; Institut de Recerca de l'Hospital Universitari de la Vall d'Hebron (VHIR) Barcelona, Spain ; Institució Catalana de Recerca i Estudis Avançats Academia Barcelona, Spain.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Soriano
keywords: 3D-reconstruction; FIB/SEM; adult neurogenesis; dendritic spines; electron microscopy; synapses