Application of the Mirror Technique for Three-Dimensional Electron Microscopy of Neurochemically Identified GABA-ergic Dendrites

Abstract

In the nervous system synaptic input arrives chiefly on dendrites and their type and distribution have been assumed pivotal in signal integration. We have developed an immunohistochemistry (IH)-correlated electron microscopy (EM) method – the “mirror” technique – by which synaptic input to entire dendrites of neurochemically identified interneurons (INs) can be mapped due preserving high-fidelity tissue ultrastructure. Hence, this approach allows quantitative assessment of morphometric parameters of synaptic inputs along the whole length of dendrites originating from the parent soma. The method exploits the fact that adjoining sections have truncated or cut cell bodies which appear on the common surfaces in a mirror fashion. In one of the sections the histochemical marker of the GABAergic subtype, calbindin was revealed in cell bodies whereas in the other section the remaining part of the very same cell bodies were subjected to serial section EM to trace and reconstruct the synaptology of entire dendrites. Here, we provide exemplary data on the synaptic coverage of two dendrites belonging to the same calbindin-D28K immunopositive IN and determine the spatial distribution of asymmetric and symmetric synapses, surface area and volume of the presynaptic boutons, morphometric parameters of synaptic vesicles, and area extent of the active zones.

Application of the mirror technique for block-face scanning electron microscopy

“…F Segmentation of pre- and postsynaptic structures across SEM images and rendering them in 3D G

Fig. 2 A Confocal image of a resin-embedded thick section processed for transmission electron microscopy (see Talapka et al 2021 ) using epi-fluorescent detection (excitation/emission at 405/461 nm). The image was taken through a coverslip under which an air-bubble inadvertently became trapped (circular area in the center).

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“…The next step was to match the block surface with the corresponding, also named as the mirror surface of the immunostained section. For this purpose, the contour of immuno-labeled neurons and nearby fiducial landmarks such as blood vessels was drawn in the sectioning plane (see Talapka et al 2021 ). To disambiguate contours of closely neighboring somata, including those of labeled and non-labeled ones, care was taken to draw all surface contours with high precision using 100 × oil objective.…”

“…Here, we introduce a solution to the above problem using a modified version of the mirror technique (Talapka et al 2021 ) that allows tracing neurochemically characterized structures in tissue blocks instead of sections. The modified technique can directly address questions regarding the distribution of synapses along entire dendrites with little or no limit in dendritic length.…”

“…Another study used the mirror technique that employed adjoining sections with cut cell bodies on the mirror surfaces. In one section, immunohistochemistry was carried out to visualize the neurochemical marker and in the other section, the complement of the cell bodies and the emerging dendrites were reconstructed using serial section TEM (Talapka et al 2021 ). While both reports managed to preserve high-quality ultrastructure, the alignment process required the use of light microscopic sections which have a limited thickness, typically, not more than 100 µm.…”

“…The spatial constraint of the above methods does not favor the full reconstruction of dendrites which often run several hundreds of µm or longer. The method presented here is an adaption of the mirror technique (Talapka et al 2021 ) which offers 3D-electron microscopic reconstruction of structures in a large tissue volume, for example, the synaptome of entire dendrites. It is based on the identification of neuronal cell bodies in the surface of thick tissue blocks using confocal reflectance imaging instead of LM of sections.…”