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J Vis Exp. 2014 Feb 12;(84):e50783. doi: 10.3791/50783.

Preparation of primary neurons for visualizing neurites in a frozen-hydrated state using cryo-electron tomography.

Author information

1
Department of Molecular Physiology and Biophysics, Baylor College of Medicine.
2
Department of Neuroscience, Baylor College of Medicine.
3
Department of Neuroscience, University of California at San Diego.
4
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine.
5
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine; wah@bcm.edu.

Abstract

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts. Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

PMID:
24561719
PMCID:
PMC4089403
DOI:
10.3791/50783
[Indexed for MEDLINE]
Free PMC Article
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