Important factors for the three-dimensional reconstruction of neuronal structures from serial ultrathin sections

Frontiers in Neural Circuits, Volume 3, Article 4

Quantitative analysis of anatomical synaptic connectivity in microcircuits depends upon accurate 3-dimentional 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 three-dimensional 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 3-dimentional reconstruction of synaptic ultrastructure. We use this methodology to reveal that parvalbumin (PV)-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.

(A, D), A synaptic contact with a cleft plane cut in parallel to the section plane is identified in the successive serial sections. The expected sequence of synapse structures is observed: (A), synaptic vesicles in the presynaptic bouton, (B), the pre-synaptic grid, (C), a postsynaptic density, and (D), the cytoplasm of the postsynaptic dendrite. (E, F), The 3D reconstructed PV dendrite (yellow) with GABA-positive (red), GABA negative (blue), and a GABA- and PV-positive bouton (pink). (E), Plot of the synaptic terminals with clearly visible synaptic clefts. (F), Plot of the same dendrite including the synaptic boutons identified by the proposed criteria. The upper surface of the dendrite also has many synaptic boutons.