Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Summary

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

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.

Introduction

Neurons establish the complex circuitry essential for the function of the central and peripheral nervous systems by elaborating dendrites to receive information and axons, often quite lengthy, to communicate with downstream neurons. Neurite outgrowth plays a fundamental role during embryonic development and neuronal differentiation and maintenance of neurites supports critically the function of the nervous system. Neuritic processes also feature critically in neuronal injury and regeneration, as well as nervous system disorders. The study of neuronal architecture is crucial to understand both the normal and diseased brain. Fortunately, physiologically relevant neurona....

Protocol

1. Preparing Dishes with EM Grids for Plating Primary Neurons

1. Examine the integrity of holey carbon on the gold EM grids using a light microscope at magnification of at least 25X. Make sure carbon holes are >98% intact.
2. For plating primary neurons, use a Bunsen burner to flame-sterilize the EM grids and concurrently render them hydrophilic. Use tweezers to pick up the EM grid by its edge, not the central gridded area. CAUTION: Never leave a lit Bunsen burner unattended, and do not use gloves or tweezers with plastic components while performing these steps:
   1. Before lighting the Bunsen burner, set the air and gas adjustments to a min....

Results

Prior to freezing and imaging via cryo-ET, light microscope images should be taken of the EM grid on which the neurons are growing. Neurites should be clearly visible without significant overlap with one another. A colored box in Figure 3A represents an area that is zoomed-in to show a higher magnification in Figure 3B, in which neurites extend across the latticework of the grid. Each grid-square is composed of a holey carbon film that supports the neurons and their neurites. These holes.......

Discussion

We show that rat embryonic neurons (dorsal root ganglion and hippocampal) can be grown on gold electron microscopy (EM) grids and frozen in vitreous ice thin enough for their neurites to be imaged using 2-D cryo-EM and 3-D cryo-ET. While hippocampal neurites have previously been imaged using cryo-ET^8,10,18,23, a protocol detailed enough for successful replication using commercially available devices has been lacking. Furthermore, while their research has pioneered the use of cryo-ET for visualizing hippocampal.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dumont #7 Tweezers	Electron Microscopy Sciences	72803-01	For handling EM grids
Glass bottom dishes	MatTek Corp.	P35G-1.5-10C	For growing sample
Electron microscopy grids	Quantifoil	Holey Carbon, Au 200, R 2/2	For growing sample
Calcium-free filter paper	Whatman	1541-055	For blotting sample
Large flat point long tweezers	Excelta Corporation	E003-000590, 25-SA	For blotting sample
Vitrification device and tweezers	FEI	Vitrobot Mark III	For freezing sample
Minigrid storage boxes	Ted Pella, Inc.	160-40	For storing EM grids
Cryo transfer holder	Gatan	626 Single Tilt Liquid Nitrogen Cryo Transfer Holder	For imaging samples
Semiautomated tilt series acquisition software	SerialEM	http://bio3d.colorado.edu/SerialEM/	For imaging samples
Image processing software	IMOD eTomo	http://bio3d.colorado.edu/imod/	For image processing
Transmission electron microscope for cryoEM	JEOL, Tokyo	200-kV JEM2100 LaB6 electron microscope	For imaging samples
4k x 4k CCD camera	Gatan	N/A	For imaging samples
3-D annotation software	Visage Imaging GmbH	Amira/Avizo	For processing 3-D data
Software for digitally stitching 2-D images	Adobe	Adobe Photoshop	For processing 2-D data
DMEM, High Glucose	Invitrogen	11965-118	For hippocampal culture
Boric acid	Sigma Aldrich	B-0252	For hippocampal culture
Sodium tetraborate	Sigma Aldrich	B-9876	For hippocampal culture
Poly-L-lysine	Sigma Aldrich	P2636-500MG	For hippocampal culture
Filter system	Corning	430758	For hippocampal culture
Neurobasal medium	Invitrogen	21103-049	For DRG culture
B-27 supplement	Invitrogen	17504-044	For DRG culture
Penicillin/Streptomycin	Invitrogen	15140-122	For DRG culture
GlutaMAX	Invitrogen	35050-061	For DRG culture
Recombinant rat b-NGF	R&D Systems	556-NG	For DRG culture
Uridine	Sigma	U3003-5G	For DRG culture
5'-Fluoro-2'-deoxyuridine	Sigma	F0503-100MG	For DRG culture
Matrigel	BD Biosciences	356234	For DRG culture