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Method Article

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

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DOI:

10.3791/50783

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February 12th, 2014

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

In This Article

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 neuronal cell culture systems exist that can recapitulate complex and heterogeneous cellular structures. Based on the elucidation of solid experimental platforms, effective visualization strategies that enable qualitative and quantitative analyses of neuronal morphology are needed. Especially useful would be a detailed methodology that provides a consistent platform for visualizing neurites, both axons and dendrites at the nanometer scale.

Traditional electron microscopy requires the use of organic solvent during dehydration and resin embedding, which can induce distortions in the specimens from their true state. To date, most structural characterizations at nanometer scale are based on larger cells or tissues that are subjected to such harsh chemicals - thus limiting the interpretation of the findings4,9,25. Moreover, for electron beam penetration, sectioning is required for organisms or cellular protrusions exhibiting a thickness greater than 1 µm 12. Finally, sectioned or milled tissue results in collection of discrete slice-specific data sets, making cumbersome the definition of the elongated feature of neurites. Even for cryo-EM, in which sectioning a frozen-hydrated specimen is possible, the method introduces compression artifacts1.

In recent years, researchers have learned how to grow hippocampal neurons directly on EM grids and flash-freezing them in liquid ethane to subsequently visualize neurites using cryo-ET8,10,18,23. However, such studies either use a custom-made device10,23, or lack details on the blotting step for generating thin enough vitreous ice for routine visualization8,18. For example, one study recommends the use of 30-40 sec for blotting the EM grid10; however, this value is optimized not for general use but is specific for that custom-made plunge-freezing device. Using a custom-made device rather than a commercially available one17 for maintaining humidity prior to plunge-freezing the sample could pose a hurdle for widespread reproducibility.

While these studies have been groundbreaking in visualizing neurites by cryo-ET, we have taken a step further to explore the applicability of cryo-ET to a variety of neuronal specimens (hippocampal and dorsal root ganglion neurons). Additionally, we discuss both optimal and suboptimal results, as well as the potential artifacts that one could encounter using cryo-ET for such specimens.

Defining a detailed technique for preserving and visualizing whole neurites at the nanometer scale in a near-native state would enhance the ability for more researchers to carry out ultrastructural studies. To this end, we describe an effective and detailed protocol using commercially available equipment to prepare unfixed, unstained neurons to visualize neurites. This is an important first step toward detailing the ultrastructure of healthy neurites and to lay the foundation for understanding what structural differences are present in nervous system disease models. Since cryo-ET can resolve unfixed, unstained neurite features in 3-D at the nanometer scale, the method will make it possible as never before to define neuritic architecture12.

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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 minimally open position.
    2. Light the Bunsen burner using a striker flint or butane lighter.
    3. Modify the air and gas adjustments to achieve a small, blue inner flame within a taller, lighter blue/violet flame. The tip of the inner flame is the hottest part of the flame. The knob underneath the burner adjusts the amount of gas entering the burner tube, while the barrel of the burner can be turned to adjust the amount of air entering the burner. Turning the air adjustment clockwise decreases the air (resulting in a purple flame) and counterclockwise increases the air (resulting in a yellow flame).
    4. Using metal tweezers (no plastic parts) to hold the EM grid, pass it quickly through the flame twice, facing carbon-side up. The carbon side will appear more matte (less shiny) and with more of a grayish tint than the other side.
    5. Immediately transfer the grid (carbon-side up) into the center of the glass-bottom dish placed within 10 cm of the Bunsen burner. Use one EM grid per dish (Figure 1). Only use glass-bottom dishes that are presterilized, i.e. via gamma irradiation.
  3. Use a light microscope to check the grid integrity (carbon holes intact) whilst still keeping the EM grid inside the glass-bottom dish (to avoid contamination). When applying any substance on the grid or anything that will come in contact with the neurons, use sterile procedure and sterile pipette tips.
  4. In a tissue culture hood using sterile procedure, apply 250 µl of the appropriate coating substance slowly and carefully to the central glass area of the Petri dish. Make sure the appropriate coating substance covers the entire EM grid.
    1. For hippocampal neurons, use poly-L-lysine (PLL, 1 mg/ml) as the coating substance, prepared as previously described19. Note that 250 µl are needed per EM grid, per dish, so scale the batch accordingly. For dorsal root ganglion (DRG) neurons, use a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (see Table of Materials and Reagents) as the coating substance, prepare as follows. Note that 250 µl are needed per EM grid, per dish, so scale the batch accordingly.
      1. Thaw a stock bottle of the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells overnight at 4 °C.
      2. Keep cold the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, and all components used to make the solution, i.e. by keeping them on ice.
      3. While on ice, dilute this gelatinous protein mixture in Neurobasal medium to yield a 1:20 dilution (i.e. dilute 500 µl of the gelatinous protein mixture into 10 ml of Neurobasal).
      4. Divide diluted gelatinous protein mixture into 1 ml aliquots, and immediately store any extra aliquots at -20 °C.
      5. Apply 250 µl per EM grid, per dish, as described in Section 1.4.
  5. Cover the Petri dish with its top and incubate (either with PLL for hippocampal specimens, OR with the gelatinous protein mixture for DRG specimens) for 1 hr at room temperature in the tissue culture hood.
  6. Aspirate all PLL (for hippocampal specimens) or the gelatinous protein mixture (for DRG specimens) from the dishes. To do this, use the vacuum system in the tissue culture hood. Use a sterile pipette tip attached to the vacuum tube. Avoid direct contact with the EM grid.
  7. Use an adjustable air-displacement pipette to carefully apply 250 µl of sterile PBS (phosphate-buffered saline) to the EM grid in the central glass area of each Petri dish, such that the EM grid is fully covered by PBS. Then, aspirate the PBS from each dish. Repeat 3x.
  8. Allow the dishes with EM grids to dry under the tissue culture hood for 15 min. Make sure they are completely dry by checking under the light microscope in the tissue culture room. Make sure there are no bubbles of moisture in the grid. If so, carefully aspirate next to the EM grid to eliminate this extra moisture. The coated grid should be used immediately for plating the neurons.

2. Preparing and Plating Primary Neurons on EM Grids

  1. To plate primary neurons, first dissect, trypsinize (using 2.5% trypsin) and triturate to dissociate the hippocampi (or dorsal root ganglion of 18-day old rat embryos) into individual cells, as previously described19.
    1. Triturate is a common term used by neurobiologists to describe dissociating clumps of cells into individual cells, particularly by use of a glass pipette with a fire-polished tip. The result is achieved by carefully and slowly drawing and releasing the cells, up and down, multiple times (~30) through the pipette.
  2. Follow procedures as previously described for DRG dissection and cell isolation24, taking note to use 0.25% trypsin (in HBSS) to isolate the rat DRG neurons, rather than use the enzymes suggested (papain, collagenase/dispase) that are more suitable for mouse DRG neurons.
  3. Calculate the number of isolated neurons (i.e. using a hemocytometer) and use this value to calculate the appropriate volume of cells to apply to each dish to achieve a concentration of 50,000 cells/ml per dish. The maximum volume to apply is 250 µl. Note that this only fills the central glass area, not the entire dish.
  4. Incubate the dishes for 30 min in a CO2 incubator at 37 °C. Allow cells to recover and adhere.
  5. Slowly add 1.5 ml of warmed media to each dish, taking care not to disturb the EM grid. The type of media depends on the cell type (hippocampal or DRG).
    1. Prepare the appropriate media for either hippocampal neurons19 or dorsal root ganglion neurons24, which is to be warmed in a 37 °C water bath prior to use. Incubate the dishes overnight in the CO2 incubator.
    2. For hippocampal neurons, change the media the next day. Change half of the media every two days onward for 14 days. Warm the media in a 37 °C water bath prior to use.
    3. For DRG neurons, the day following dissection/plating, prepare a fresh stock of media with an anti-mitotic agent (Uridine and 5'-Fluoro-2'-deoxyuridine) make a 10 mM stock solution of each, separately; use a final concentration of 10 µM for each. Remove half of the DRG media (875 µl) and add 875 µl of fresh media with the anti-mitotic agent.
      1. For DRG neurons, every two days for the first week, alternate changing media between anti-mitotic media and standard DRG media. For the second week, change standard DRG media every two days.

3. Vitrifying Neurons on EM Grids

  1. Prepare equipment and all materials for freezing and storing the gold EM grids at cryogenic temperature: a vitrification device with a humidity chamber17, fine-point specialized tweezers for the vitrification machine, long flat point tweezers, dewar(s) of liquid nitrogen (LN2), coolant container, EM grid storage box, calcium-free filter paper.
  2. Start the vitrification device. Set the humidity to 100% and temperature to 32 °C. In the “Console” section, set the blot time to zero seconds. This allows for manual blotting through the side-window of the vitrification machine's humidity chamber.
  3. Handle the calcium-free filter paper with gloves, layering them such that three papers are stacked. Cut the stack into 0.5 cm wide strips that are ~2 cm long. Bend them at a 90° angle such that one side of the paper has a 0.5 cm x 0.5 cm face (Figure 2). Using tweezers, remove the middle paper and place it on another calcium-free filter paper until use.
  4. Put the grid storage button in the button holder within the vitrification chamber. Fill the inner chamber of the coolant container with liquid nitrogen and wait until complete evaporation. Fill the outer chamber of the coolant container with liquid nitrogen until it attains stable temperature for proceeding to the next step. Fill the inner chamber with high-purity gaseous ethane that will condense to a liquid state within the cooled chamber.
  5. Move the dish(es) from the incubator to a large 100 mm polystyrene dish for transport to the vitrification room, if not within the immediate vicinity. Use the specialized vitrification tweezers to carefully pick the EM grid from the dish. Note which side the neurons are growing on the EM grid; the position will matter for the next step. Use the black sliding lock on the tweezers to securely lock the tweezers on the EM grid.
  6. Insert the tweezers into the vitrification machine such that side of the EM grid on which the neurons are adhered faces to the left, away from the side-opening hole of the vitrification machine. Retract the specialized tweezers into the vitrification machine.
  7. Place the coolant container in the appropriate holder of the vitrification machine. It should be filled with adequate LN2 and liquid ethane. Using the appropriate screen command of the vitrification machine, raise the coolant chamber upward until it's flushed with the bottom of the humidity chamber.
  8. With the flat point tweezers, grasp one edge of the filter paper such that the shorter side (the 0.5 cm x 0.5 cm face) is perpendicular to the tweezers. This face will come into direct contact with the EM grid for blotting (Figure 2B). Carefully insert the filter paper into the side-hole of the vitrification machine's humidity chamber (Figure 2A). Stably hold the paper against the EM grid (the side facing away from the specimen) for 10 sec. Discard the paper afterward and immediately plunge-freeze the specimen in the liquid ethane using the vitrification machine's automation.
  9. Carefully transfer your frozen-hydrated EM grid to one of the slots in one of the grid storage buttons. Repeat the process for additional EM grids in the dishes. The EM grid storage buttons used in these experiments can store multiple frozen-hydrated EM grids.

4. Image Collection, Processing, and Annotation

  1. Proceed to collect 2-D electron micrographs and/or 3-D tilt series5 of the neurites using a cryo-electron microscope under a low-dose condition. The 2-D images were intended to assess the quality of the grid in terms of ice thickness and the possible areas to be useful for 3-D tilt series.
    1. In this case, all images were collected using a 4k x 4k CCD camera attached to a 200 kV electron microscope equipped with a single tilt liquid nitrogen cryo transfer holder, at 20k microscope magnification at a target underfocus of 7 µm and sampling of 4.4 Å/pixel.
    2. To obtain a 3D tomogram of the sample, take a series of projection images while incrementally tilting the sample along one axis of the transmission electron microscope (TEM). Given the tilt angles and other experimental settings, there are several different softwares available to automatically collect the tilt series5. The 3-D tilt series shown here were collected on the same microscope using semi-automated tilt series acquisition software 26 over a range of -60° to 60° at 5° increments with a cumulative dose of ~60 e/Å2.
  2. Reconstruct the tilt series of the neurites using image processing software21 as previously described for other samples5,29.
  3. Color-annotate the 3-D features of the neurites by first segmenting the tomogram and creating a surface model using a 3-D image processing software as previously described5.

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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...

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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-ET8,10,18,23, 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...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

This research has been supported by the NIH grants (PN2EY016525 and P41GM103832). S.H.S. was supported by a fellowship from the Nanobiology Interdisciplinary Graduate Training Program of the W. M. Keck Center for Interdisciplinary Bioscience Training of the Gulf Coast Consortia (NIH Grant No. T32EB009379).

S.H.S. dissected, grew and vitrified DRG and hippocampal cells; collected cryo-EM and cryo-ET data of DRG and hippocampal axons; reconstructed and color-annotated the tilt series. M.R.G. dissected and provided hippocampal cells in M.N.R.’s lab. S.C. assisted in tilt series annotation. S.H.S. was trained by C.W. on how to dissect and grow neurons. S.H.S., W.C.M. and W.C. conceived the experiments. S.H.S. prepared the manuscript with input from other authors.

This video was filmed at the Center for Cellular Imaging and NanoAnalytics (C-CINA) of the Biozentrum of the University Basel. C-CINA is integrated into the Department for Biosystems Science and Engineering (D-BSSE) of the ETH Zürich, located in Basel, Switzerland.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Dumont #7 TweezersElectron Microscopy Sciences72803-01For handling EM grids
Glass bottom dishesMatTek Corp.P35G-1.5-10CFor growing sample
Electron microscopy gridsQuantifoilHoley Carbon, Au 200, R 2/2For growing sample
Calcium-free filter paperWhatman1541-055For blotting sample
Large flat point long tweezersExcelta CorporationE003-000590, 25-SAFor blotting sample 
Vitrification device and tweezersFEIVitrobot Mark IIIFor freezing sample
Minigrid storage boxesTed Pella, Inc.160-40For storing EM grids
Cryo transfer holderGatan626 Single Tilt Liquid Nitrogen Cryo Transfer HolderFor imaging samples
Semiautomated tilt series acquisition softwareSerialEMhttp://bio3d.colorado.edu/SerialEM/For imaging samples
Image processing softwareIMOD eTomohttp://bio3d.colorado.edu/imod/For image processing
Transmission electron microscope for cryoEMJEOL, Tokyo200-kV JEM2100 LaB6 electron microscopeFor imaging samples
4k x 4k CCD cameraGatanN/AFor imaging samples
3-D annotation softwareVisage Imaging GmbHAmira/AvizoFor processing 3-D data
Software for digitally stitching 2-D imagesAdobeAdobe PhotoshopFor processing 2-D data
DMEM, High GlucoseInvitrogen11965-118For hippocampal culture
Boric acidSigma AldrichB-0252For hippocampal culture
Sodium tetraborateSigma AldrichB-9876For hippocampal culture
Poly-L-lysine Sigma AldrichP2636-500MGFor hippocampal culture
Filter systemCorning430758For hippocampal culture
Neurobasal mediumInvitrogen21103-049For DRG culture
B-27 supplementInvitrogen17504-044For DRG culture
Penicillin/StreptomycinInvitrogen15140-122For DRG culture
GlutaMAXInvitrogen35050-061For DRG culture
Recombinant rat b-NGFR&D Systems556-NGFor DRG culture
UridineSigmaU3003-5GFor DRG culture
5'-Fluoro-2'-deoxyuridineSigmaF0503-100MGFor DRG culture
MatrigelBD Biosciences356234For DRG culture

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Neurite VisualizationElectron Microscopy GridsVitrification ProcedureHippocampal NeuronsDorsal Root GanglionNanometer Resolution3D Reconstruction
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
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