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.&#8217;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 Z&#252;rich, located in Basel, Switzerland.

1. Preparing Dishes with EM Grids for Plating Primary Neurons
<ol>
	<li>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 &#62;98% intact.</li>
	<li>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 ...

<ol>
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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...

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 &#181;m 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.

<table border="0" cellpadding="0" cellspacing="0" width="970">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #7 Tweezers</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:309px;">72803-01</td>
			<td style="width:171px;">For handling EM grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom dishes</td>
			<td>MatTek Corp.</td>
			<td>P35G-1.5-10C</td>
			<td>For growing sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electron microscopy grids</td>
			<td>Quantifoil</td>
			<td>Holey Carbon, Au 200, R 2/2</td>
			<td>For growing sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium-free filter paper</td>
			<td>Whatman</td>
			<td>1541-055</td>
			<td>For blotting sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large flat point long tweezers</td>
			<td>Excelta Corporation</td>
			<td>E003-000590, 25-SA</td>
			<td>For blotting sample </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vitrification device and tweezers</td>
			<td>FEI</td>
			<td>Vitrobot Mark III</td>
			<td>For freezing sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini grid storage boxes</td>
			<td>Ted Pella, Inc.</td>
			<td>160-40</td>
			<td>For storing EM grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryo transfer holder</td>
			<td>Gatan</td>
			<td>626 Single Tilt Liquid Nitrogen Cryo Transfer Holder</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Semi-automated tilt series acquisition software</td>
			<td>SerialEM</td>
			<td>http://bio3d.colorado.edu/SerialEM/</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image processing software</td>
			<td>IMOD eTomo</td>
			<td>http://bio3d.colorado.edu/imod/</td>
			<td>For image processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Transmission electron microscope for cryoEM</td>
			<td>JEOL, Tokyo</td>
			<td>200-kV JEM2100 LaB6 electron microscope</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4k x 4k CCD camera</td>
			<td>Gatan</td>
			<td>N/A</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-D annotation software</td>
			<td>Visage Imaging GmbH</td>
			<td>Amira/Avizo</td>
			<td>For processing 3-D data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software for digitally stitching 2-D images</td>
			<td>Adobe</td>
			<td>Adobe Photoshop</td>
			<td>For processing 2-D data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, High Glucose</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B-0252</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium tetraborate</td>
			<td>Sigma Aldrich</td>
			<td>B-9876</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine </td>
			<td>Sigma Aldrich</td>
			<td>P2636-500MG</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter system</td>
			<td>Corning</td>
			<td>430758</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant rat b-NGF</td>
			<td>R&#38;D Systems</td>
			<td>556-NG</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine</td>
			<td>Sigma</td>
			<td>U3003-5G</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5&#39;-Fluoro-2&#39;-deoxyuridine</td>
			<td>Sigma</td>
			<td>F0503-100MG</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>For DRG culture</td>
		</tr>
	</tbody>
</table>

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

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.

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...

Watch this Scientific Journal Video about Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography at JoVE.com

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

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.

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

preparation-primary-neurons-for-visualizing-neurites-frozen-hydrated

Research

JoVE Journal

Neuroscience

79.0K visualizações.  Baylor College of Medicine.  Para preservar processos neuronais para análise ultra-estrutural, nós descrevemos um protocolo para revestimento dos neurônios primários em grades de microscopia eletrônica seguido de congelamento flash, rendendo amostras suspensas em uma camada de gelo vítreo. Essas amostras podem ser examinadas com um microscópio crio-eletrônico para visualizar estruturas em escala nanométrica. 