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

Sarah H. Shahmoradian, Mauricio R. Galiano, Chengbiao Wu, Shurui Chen, Matthew N. Rasband, William C. Mobley, Wah Chiu

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

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.

Original languageEnglish (US)
Article numbere50783
JournalJournal of Visualized Experiments
Issue number84
DOIs
StatePublished - Feb 12 2014
Externally publishedYes

Keywords

  • Brain
  • Cryo-electron microscopy
  • Electron microscope Tomography
  • Issue 84
  • Morphological assay
  • Neurons
  • Neuroscience
  • Primary neuron culture
  • Rat

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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