Top

Published in:

Open Access 28-05-2022 | Methods Paper

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

Authors: Petra Talapka, Bence Béla Bába, Zoltán Mészár, Réka Eszter Kisvárday, Zsolt Kocsis, Mohit Srivastava, Zoltán Kisvárday

Published in: Brain Structure and Function | Issue 6/2022

Abstract

The mirror technique adapted for electron microscopy allows correlating neuronal structures across the cutting plane of adjoining light microscopic sections which, however, have a limited thickness, typically less than 100 µm (Talapka et al. in Front Neuroanat, 2021, https://doi.org/10.3389/fnana.2021.652422). Here, we extend the mirror technique for tissue blocks in the millimeter range and demonstrate compatibility with serial block-face electron microscopy (SBEM). An essential step of the methodological improvement regards the recognition that unbound resin must be removed from the tissue surface to gain visibility of surface structures. To this, the tissue block was placed on absorbent paper during the curing process. In this way, neuronal cell bodies could be unequivocally identified using epi-illumination and confocal microscopy. Thus, the layout of cell bodies which were cut by the sectioning plane can be correlated with the layout of their complementary part in the adjoining section processed for immunohistochemistry. The modified mirror technique obviates the spatial limit in investigating synaptology of neurochemically identified structures such as neuronal processes, dendrites and axons.

Available only for authorised users

Besser S, Sicker M, Marx G, Winkler U, Eulenburg V et al (2015) A transgenic mouse line expressing the red fluorescent protein tdTomato in GABaergic neurons. PLoS ONE 10(6):e0129934. https://doi.org/10.1371/journal.pone.0129934CrossRefPubMedPubMedCentral

Bock DD, Lee WC, Kerlin AM, Andermann ML, Hood G, Wetzel AW et al (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471:177–182. https://doi.org/10.1038/nature09802CrossRefPubMedPubMedCentral

Briggman KL, Bock DD (2012) Volume electron microscopy for neuronal circuit reconstruction. Curr Opin Neurobiol 22:154–161. https://doi.org/10.1016/j.conb.2011.10.022CrossRefPubMed

Colonnier M (1968) Synaptic pattern on different cell types in the different laminae of the cat visual cortex. Electron Microsc Stud Brain Res 9(2):268–287

Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329. https://doi.org/10.1371/journal.pbio.0020329CrossRefPubMedPubMedCentral

Fishell G, Kepecs A (2020) Interneuron types as attractors and controllers. Annu Rev Neurosci 43:1–30. https://doi.org/10.1146/annurev-neuro-070918-050421CrossRefPubMed

Goetz L, Roth A, Häusser M (2021) Active dendrites enable strong but sparse inputs to determine orientation selectivity. PNAS 118(30):e2017339118. https://doi.org/10.1073/pnas.2017339118CrossRefPubMedPubMedCentral

Gray EG (1959) Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. J Anat (lond) 93:420–433

He M, Tucciarone J, Lee S, Nigro MJ, Kim Y, Levine JM, Kelly SM, Krugikov I, Wu P, Chen Y et al (2016) Strategies and tools for combinatorial targeting of GABAergic neurons in mouse cerebral cortex. Neuron 91:1228–1243CrossRef

Helmstaedter M (2013) Cellular-resolution connectomics: challenges of dense neural circuit reconstruction. Nat Methods 10:501–507. https://doi.org/10.1038/nmeth.2476CrossRefPubMed

Hu H, Vervaeke K (2018) Synaptic integration in cortical inhibitory neuron dendrites. Neurosci 368:115–131CrossRef

Jin X, Mathers PH, Szabo G, Katarova Z, Agmon A (2001) vertical bias in dendritic trees of non-pyramidal neocortical neurons expressing GAD67–GFP in vitro. Cereb Cortex 11(7):666–678. https://doi.org/10.1093/cercor/11.7.666CrossRefPubMed

Kanari L, Ramaswamy S, Shi Y, Morand S, Meystre J, Perin R, Abdellah M, Wang Y, Hess K, Markram H (2019) Objective morphological classification of neocortical pyramidal cells. Cereb Cortex 29(4):1719–1735. https://doi.org/10.1093/cercor/bhy339CrossRefPubMedPubMedCentral

Kirchner JH, Gjorgjieva J (2021) Emergence of local and global synaptic organization on cortical dendrites. Nat Commun 12:4005. https://doi.org/10.1038/s41467-021-23557-3CrossRefPubMedPubMedCentral

Knott GW, Holtmaat A, Trachtenberg JT, Svoboda K, Welker E (2009) A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nat Protoc 4:1145–1156. https://doi.org/10.1038/nprot.2009.114CrossRefPubMed

Kubota Y, Sohn J, Hatada S, Schurr M, Straehle J, Gour A, Neujahr R, Miki T, Mikula S, Kawaguchi Y (2018) A carbon nanotube tape for serial-section electron microscopy of brain ultrastructure. Nat Commun 9:437. https://doi.org/10.1038/s41467-017-02768-7CrossRefPubMedPubMedCentral

Lein ES, Hawrylycz MJ, Ao N, Ayres M, Besinger A, Bernard A et al (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445(7124):168–176. https://doi.org/10.1038/nature05453CrossRefPubMed

Maclachlan C, Sahlender DA, Hayashi S, Molnár Z, Knott G (2018) Block face scanning electron microscopy of fluorescently labeled axons without using near infra-red branding. Front Neuroanat 12:88. https://doi.org/10.3389/fnana.2018.00088CrossRefPubMedPubMedCentral

Micheva KD, Smith SJ (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55:25–36. https://doi.org/10.1016/j.neuron.2007.06.014CrossRefPubMedPubMedCentral

Poirazi P, Papoutsi A (2020) Illuminating dendritic function with computational models. Nat Rev Neurosci 21(6):303–321. https://doi.org/10.1038/s41583-020-0301-7CrossRefPubMed

Prönneke A, Scheuer B, Wagener RJ, Möck M, Witte M, Staiger JF (2015) Characterizing VIP neurons in the barrel cortex of VIPcre/tdTomato mice reveals layer-specific differences. Cereb Cortex 25(12):4854–4868. https://doi.org/10.1093/cercor/bhv202CrossRefPubMedPubMedCentral

Rees CL, White CM, Ascoli GA (2017) Neurochemical markers in the mammalian brain: structure, roles in synaptic communication, and pharmacological relevance. Current Med Chem 24(28):3077–3103. https://doi.org/10.2174/0929867324666170414163506CrossRef

Shepherd GM, Brayton RK, Segev MJP, I, Rinzel j, Rall W, (1985) Signal enhancement in distal cortical dendrites by means of interactions between active dendritic spines. PNAS 82(7):2192–2195. https://doi.org/10.1073/pnas.82.7.2192CrossRefPubMedPubMedCentral

Sohn J, Okamoto S, Kataoka N, Kaneko T, Nakamura K, Hioki H (2016) Differential inputs to the perisomatic and distal-dendritic compartments of VIP-positive neurons in layer 2/3 of the mouse barrel cortex. Front Neuroanat 10:124. https://doi.org/10.3389/fnana.2016.00124CrossRefPubMedPubMedCentral

Talapka P, Zs K, Marsi LD, Szarvas VE, Kisvárday ZF (2021) Application of the mirror technique for three-dimensional electron microscopy of neurochemically identified GABA-ergic dendrites. Front Neuroanat. https://doi.org/10.3389/fnana.2021.652422CrossRefPubMedPubMedCentral

Tapia JC, Kasthuri N, Hayworth K, Schalek R, Lichtman JW, Smith SJ, Buchanan J (2012) High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. Nat Protoc 7:193–206. https://doi.org/10.1038/nprot.2011.439CrossRefPubMedPubMedCentral

Tasic B, Menon V, Nguyen T et al (2016) Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat Neurosci 19:335–346. https://doi.org/10.1038/nn.4216CrossRefPubMedPubMedCentral

Titze B, Genoud C, Friedrich RW (2018) SBEMimage: versatile acquisition control software for serial block-face electron microscopy. Front Neural Circuits 12:24. https://doi.org/10.3389/fncir.2018.00054CrossRef

Tzilivaki A, Kastellakis G, Poirazi P (2019) Challenging the point neuron dogma: fs basket cells as 2-stage nonlinear integrators. Nat Commun 10(1):1–14. https://doi.org/10.1038/s41467-019-11537-7CrossRef

Zeisel A, Muňoz-Manchado AB, Codeluppi S et al (2015) Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347:1138–1142. https://doi.org/10.1126/science.aaa1934CrossRefPubMed

Title: Application of the mirror technique for block-face scanning electron microscopy
Authors: Petra Talapka
Bence Béla Bába
Zoltán Mészár
Réka Eszter Kisvárday
Zsolt Kocsis
Mohit Srivastava
Zoltán Kisvárday
Publication date: 28-05-2022
Publisher: Springer Berlin Heidelberg
Published in: Brain Structure and Function / Issue 6/2022
Print ISSN: 1863-2653
Electronic ISSN: 1863-2661
DOI: https://doi.org/10.1007/s00429-022-02506-w

ACC 2024 Congress

Springer Medicine

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

Abstract

ACC 2024 Congress

Springer Medicine

Abstract

Please log in to get access to this content

Other articles of this Issue 6/2022

White matter tract conductivity is resistant to wide variations in paranodal structure and myelin thickness accompanying the loss of Tyro3: an experimental and simulated analysis

The PV2 cluster of parvalbumin neurons in the murine periaqueductal gray: connections and gene expression

Correction to: Tracing in vivo the dorsal loop of the optic radiation: convergent perspectives from tractography and electrophysiology compared to a neuroanatomical ground truth

Individual variability in the nonlinear development of the corpus callosum during infancy and toddlerhood: a longitudinal MRI analysis

Aberrant topological organization and age-related differences in the human connectome in subjective cognitive decline by using regional morphology from magnetic resonance imaging

Neuronal nitric oxyde synthase positive neurons in human indusium griseum