TY - JOUR
T1 - FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons
AU - Bosch, Carles
AU - Martínez, Albert
AU - Masachs, Nuria
AU - Teixeira, Cátia M.
AU - Fernaud, Isabel
AU - Ulloa, Fausto
AU - Pérez-Martínez, Esther
AU - Lois, Carlos
AU - Comella, Joan X.
AU - De-Felipe, Javier
AU - Merchán-Pérez, Angel
AU - Soriano, Eduardo
PY - 2015/5/21
Y1 - 2015/5/21
N2 - © 2015 Bosch, Martínez, Masachs, Teixeira, Fernaud, Ulloa, Pérez-Martínez, Lois, Comella, DeFelipe, Merchán-Pérez and Soriano. 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.
AB - © 2015 Bosch, Martínez, Masachs, Teixeira, Fernaud, Ulloa, Pérez-Martínez, Lois, Comella, DeFelipe, Merchán-Pérez and Soriano. 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.
KW - 3D-reconstruction
KW - Adult neurogenesis
KW - Dendritic spines
KW - Electron microscopy
KW - FIB/SEM
KW - Synapses
U2 - 10.3389/fnana.2015.00060
DO - 10.3389/fnana.2015.00060
M3 - Article
SN - 1662-5129
VL - 9
JO - Frontiers in Neuroanatomy
JF - Frontiers in Neuroanatomy
M1 - 60
ER -