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Front Neuroanat. 2015 May 21;9:60. doi: 10.3389/fnana.2015.00060. eCollection 2015.

FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons.

Author information

1
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.
2
Developmental Neurobiology and Regeneration Unit, Department of Cell Biology and Parc Cientific de Barcelona, University of Barcelona Barcelona, Spain.
3
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.
4
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.
5
Department of Neurobiology, University of Massachusetts Medical School Worcester, MA, USA.
6
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.
7
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.
8
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.

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.

KEYWORDS:

3D-reconstruction; FIB/SEM; adult neurogenesis; dendritic spines; electron microscopy; synapses

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