Tissue Multicolor STED Nanoscopy of Presynaptic Proteins in the Calyx of Held

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Abstract

The calyx of Held, a large glutamatergic terminal in the mammalian auditory brainstem has been extensively employed to study presynaptic structure and function in the central nervous system. Nevertheless, the nanoarchitecture of presynaptic proteins and subcellular components in the calyx terminal and its relation to functional properties of synaptic transmission is only poorly understood. Here, we use stimulated emission depletion (STED) nanoscopy of calyces in thin sections of aldehyde-fixed rat brain tissue to visualize immuno-labeled synaptic proteins including VGluT1, synaptophysin, Rab3A and synapsin with a lateral resolution of approximately 40 nm. Excitation multiplexing of suitable fluorescent dyes deciphered the spatial arrangement of the presynaptic phospho-protein synapsin relative to synaptic vesicles labeled with anti-VGluT1. Both predominantly occupied the same focal volume, yet may exist in exclusive domains containing either VGluT1 or synapsin immunoreactivity. While the latter have been observed with diffraction-limited fluorescence microscopy, STED microscopy for the first time revealed VGluT1-positive domains lacking synapsins. This observation supports the hypothesis that molecularly and structurally distinct synaptic vesicle pools operate in presynaptic nerve terminals.

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The synapsins: key actors of synapse function and plasticity.

F Cesca, P. Baldelli, F Valtorta … (2010)

The synapsins are a family of neuronal phosphoproteins evolutionarily conserved in invertebrate and vertebrate organisms. Their best-characterised function is to modulate neurotransmitter release at the pre-synaptic terminal, by reversibly tethering synaptic vesicles (SVs) to the actin cytoskeleton. However, many recent data have suggested novel functions for synapsins in other aspects of the pre-synaptic physiology, such as SV docking, fusion and recycling. Synapsin activity is tightly regulated by several protein kinases and phosphatases, which modulate the association of synapsins to SVs as well as their interaction with actin filaments and other synaptic proteins. In this context, synapsins act as a link between extracellular stimuli and the intracellular signalling events activated upon neuronal stimulation. Genetic manipulation of synapsins in various in vivo models has revealed that, although not essential for the basic development and functioning of neuronal networks, these proteins are extremely important in the fine-tuning of neuronal plasticity, as shown by the epileptic phenotype and behavioural abnormalities characterising mouse lines lacking one or more synapsin isoforms. In this review, we summarise the current knowledge about how the various members of the synapsin family are involved in the modulation of the pre-synaptic physiology. We give a comprehensive description of the molecular basis of synapsin function, as well as an overview of the more recent evidence linking mutations in the synapsin proteins to the onset of severe central nervous system diseases such as epilepsy and schizophrenia. (c) 2010 Elsevier Ltd. All rights reserved.

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2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.

Johann Engelhardt, T Staudt, Stefanie Lang … (2006)

The use of high numerical aperture immersion lenses in optical microscopy is compromised by spherical aberrations induced by the refractive index mismatch between the immersion system and the embedding medium of the sample. Especially when imaging >10 micro m deep into the specimen, the refractive index mismatch results in a noticeable loss of image brightness and resolution. A solution to this problem is to adapt the index of the embedding medium to that of the immersion system. Unfortunately, not many mounting media are known that are both index tunable as well as compatible with fluorescence imaging. Here we introduce a nontoxic embedding medium, 2,2'-thiodiethanol (TDE), which, by being miscible with water at any ratio, allows fine adjustment of the average refractive index of the sample ranging from that of water (1.33) to that of immersion oil (1.52). TDE thus enables high resolution imaging deep inside fixed specimens with objective lenses of the highest available aperture angles and has the potential to render glycerol embedding redundant. The refractive index changes due to larger cellular structures, such as nuclei, are largely compensated. Additionally, as an antioxidant, TDE preserves the fluorescence quantum yield of most of the fluorophores. We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures.

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Three-dimensional reconstruction of a calyx of Held and its postsynaptic principal neuron in the medial nucleus of the trapezoid body.

Kurt Sätzler, Leander F Söhl, Johann H Bollmann … (2002)

The three-dimensional morphology of the axosomatic synaptic structures between a calyx of Held and a principal neuron in the medial nucleus of the trapezoid body (MNTB) in the brainstem of young postnatal day 9 rats was reconstructed from serial ultrathin sections. In the apposition zone between the calyx and the principal neuron two types of membrane specializations were identified: synaptic contacts (SCs) with active zones (AZs) and their associated postsynaptic densities (PSDs) constituted approximately 35% (n = 554) of the specializations; the remaining 65% (n = 1010) were puncta adherentia (PA). Synaptic contacts comprised approximately 5% of the apposition area of presynaptic and postsynaptic membranes. A SC had an average area of 0.100 microm(2), and the nearest neighbors were separated, on average, by 0.59 microm. Approximately one-half of the synaptic vesicles in the calyx were clustered within a distance of 200 nm of the AZ membrane area, a cluster consisting of approximately 60 synaptic vesicles (n = 52 SCs). Approximately two synaptic vesicles per SC were "anatomically docked." Comparing the geometry of the synaptic structure with its previously studied functional properties, we find that during a single presynaptic action potential (AP) (1) approximately 35% of the AZs release a transmitter quantum, (2) the number of SCs and anatomically docked vesicles is comparable with the low estimates of the readily releasable pool (RRP) of quanta, and (3) the broad distribution of PSD areas [coefficient of variation (CV) = 0.9] is likely to contribute to the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and rising phase of the EPSC during a single AP.

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Author and article information

Contributors

Jean-Pierre Mothet: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2013

Publication date (Electronic): 26 April 2013

Volume: 8

Issue: 4

Electronic Location Identifier: e62893

Affiliations

[1 ]Institute of Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany

[2 ]Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

[3 ]Bioquant Center, University of Heidelberg, Heidelberg, Germany

[4 ]Optical Nanoscopy Division, German Cancer Research Center (DKFZ), Heidelberg, Germany

CNRS - Université Aix Marseille, France

Author notes

* E-mail: kuner@ 123456uni-heidelberg.de

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: TK SWH JE. Performed the experiments: CK TS PB HH. Analyzed the data: CK TS PB JE TK. Wrote the paper: CK TK.

Article

Publisher ID: PONE-D-12-33419

DOI: 10.1371/journal.pone.0062893

PMC ID: 3637247

PubMed ID: 23658655

SO-VID: 8b7fe70e-887c-4331-b18b-9ab984654306

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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 30 October 2012

Date accepted : 27 March 2013

Page count

Pages: 10

Funding

This work has been partially supported by the CellNetworks Cluster of Excellence (EXC81, T.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.

Tissue Multicolor STED Nanoscopy of Presynaptic Proteins in the Calyx of Held

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Most cited references 11

The synapsins: key actors of synapse function and plasticity.

2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.

Three-dimensional reconstruction of a calyx of Held and its postsynaptic principal neuron in the medial nucleus of the trapezoid body.

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