Abstract
Quantitative analysis of anatomical synaptic connectivity in microcircuits depends upon accurate three-dimensional (3D) reconstructions of synaptic ultrastructure using electron microscopy of serial ultrathin sections. Here we address two pitfalls in current methodology that lead to inaccurate reconstructions and compromise conclusions drawn from the data. The first pitfall is inaccurate determination of ultrathin section thickness, which negatively affects the 3D shape of reconstructions and therefore impairs quantitative measurement of synaptic structures. Secondly, current methodology significantly underestimates the number of synaptic junctions, with only two-thirds or less of genuine synaptic contacts being identified in dendrites that radiate within the plane of section. Here we propose a new methodology utilizing precise optical measurements of section thickness and successive observations of synaptic elements across serial ultrathin sections that corrects for these limitations to allow accurate 3D reconstruction of synaptic ultrastructure. We use this methodology to reveal that parvalbumin-expressing cortical interneurons have a much higher synaptic density than previously shown. This result suggests that this technique will be useful for re-examining synaptic connectivity of other cell types.
Highlights
A deep understanding of cortical microcircuitry requires accurate quantitative measurements of morphological parameters
The sections were incubated in 0.1 M phosphate buffer (PB) containing 1% sodium borohydrate for 30 min and in 0.05 M Tris-buffered saline (TBS) containing 1% H2O2 for 30 min before incubation with primary antiserum against parvalbumin developed in mouse (1:4000, P-3171, Sigma-Aldrich, Saint Louis, MO, USA) diluted in TBS containing 10% normal goat serum and 2% bovine serum albumin overnight at 4°C
We propose two new methodologies that improve the accuracy of 3D reconstruction of neuronal ultrastructure and facilitate accurate synapse identification
Summary
A deep understanding of cortical microcircuitry requires accurate quantitative measurements of morphological parameters Such measurements are reliably made from three-dimensional (3D) reconstructions of tissue structure using electron-microscopic (EM) images of successive ultrathin sections (Fiala et al, 2002; Holtmaat et al, 2006; Karube et al, 2004; Kubota and Kawaguchi, 2000; Kubota et al, 2007; White et al, 1994). Section thickness is assumed to be one-half the width of the ridges This method generates results that vary according to the condition of the resin, the type of tissue embedded, and the height of the fold. We introduce a new methodology that overcomes these limitations allowing accurate morphological reconstruction of neuronal tissue and identification of synaptic contacts occurring at all angles relative to the plane of section
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