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. 2011 Dec 21;2(12):700-4.
doi: 10.1021/cn200094j. Epub 2011 Oct 4.

Label-free visualization of ultrastructural features of artificial synapses via cryo-EM

Gopakumar Gopalakrishnan  1 , Patricia T Yam, Carolin Madwar, Mihnea Bostina, Isabelle Rouiller, David R Colman, R Bruce Lennox
Affiliations

Label-free visualization of ultrastructural features of artificial synapses via cryo-EM

Gopakumar Gopalakrishnan et al. ACS Chem Neurosci. .

Abstract

The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryo-electron microscopy (cryo-EM). The silica beads are derivatized by poly-d-lysine or lipid bilayers. Molecular features known to exist at presynapses are clearly present at these artificial synapses, as visualized by cryo-EM. Key synaptic features such as the membrane contact area at synaptic junctions, the presynaptic bouton containing presynaptic vesicles, as well as microtubular structures can be identified. This is the first report of the direct, label-free observation of ultrastructural details of artificial synapses.

Keywords: Artificial synapses; cryo-EM; electron microscopy; hippocampal neurons; synaptic vesicles.

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Figures

Scheme 1
Scheme 1. Scheme (not to scale) Illustrating the Experimental Steps
Isolated hippocampal neurons are plated onto sterile, PLL coated Au/Quantifoil EM grids. These cells are cultured for 7 days or more prior to cryo-EM imaging.
Figure 1
Figure 1
Representative cryo-EM images showing hippocampal neurons (DIV 8) grown on a sterile, PLL-coated Au/Quantifoil grid. (A) Neuronal processes are visible along the grids, which when on the holes (black arrows) grow a little wider (white arrows) than on the support portion of the grid. The black arrowheads indicate the areas where the axons grow slightly wider outside the holes. Two axonal processes are highlighted (white dots) to indicate the large length scales they are grown on the grid. (B) Magnified view of one of the holes where synapses (white arrows) from closely associated neural processes are visible. High electron density is observed at the areas where synaptic vesicles have accumulated (black arrows). The inset in (B) shows synaptic vesicles observed at the presynaptic boutons.
Figure 2
Figure 2
Representative cryo-EM image (A) showing an artificial synapse formed between hippocampal neurons (DIV 8) grown on a sterile, PLL coated Au/Quantifoil grid and poly-d-lysine coated 500 nm silica beads. Microtubular networks (white arrow heads) are also visible. Similarly coated beads that are not in contact with neurons are also visible in the image. Few synaptic vesicles (black arrowheads) are also visible. White arrow indicates the contact area at the synaptic junction, which in this case is between an axon and a bead. (B) Simplified sketch (not to scale) depicting the structural components of the artificial synapse seen in (A).
Figure 3
Figure 3
Representative cryo-EM image (A) showing an artificial synapse formed between hippocampal neurons (DIV 8) grown on a sterile, PLL coated Au/Quantifoil grid and poly-d-lysine coated 500 nm silica bead. Only part of the bead is seen in this image. The white arrow indicates the contact area at the synaptic junction. Synaptic vesicles are more clearly visualized (white arrowheads) in this image as compared to Figure 1. The larger vesicles (black arrowheads) could be the transport vesicles. Black arrows show the direction of the axonal shaft growth. (B) Simplified sketch (not to scale) depicting the structural components of the artificial synapse seen in (A).
See this image and copyright information in PMC

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