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 tweezers with plastic components while performing these steps:
	<ol>
		<li>Before lighting the Bunsen burner, set the air and gas adjustments to a minimally open position.</li>
		<li>Light the Bunsen burner using a striker flint or butane lighter.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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&#160;1). Only use glass-bottom dishes that are presterilized, i.e. via gamma irradiation.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>In a tissue culture hood using sterile procedure, apply 250 &#181;l 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.
	<ol>
		<li>For hippocampal neurons, use poly-L-lysine (PLL, 1 mg/ml) as the coating substance, prepared as previously described19. Note that 250 &#181;l 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 &#181;l are needed per EM grid, per dish, so scale the batch accordingly.
		<ol>
			<li>Thaw a stock bottle of the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells overnight at 4 &#176;C.</li>
			<li>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.</li>
			<li>While on ice, dilute this gelatinous protein mixture in Neurobasal medium to yield a 1:20 dilution (i.e.&#160;dilute 500 &#181;l of the gelatinous protein mixture into 10 ml of Neurobasal).</li>
			<li>Divide diluted gelatinous protein mixture into 1 ml aliquots, and immediately store any extra aliquots at -20 &#176;C.</li>
			<li>Apply 250 &#181;l per EM grid, per dish, as described in Section 1.4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use an adjustable air-displacement pipette to carefully apply 250 &#181;l 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.</li>
	<li>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.</li>
</ol>
2. Preparing and Plating Primary Neurons on EM Grids
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#181;l. Note that this only fills the central glass area, not the entire dish.</li>
	<li>Incubate the dishes for 30 min in a CO2 incubator at 37 &#176;C. Allow cells to recover and adhere.</li>
	<li>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).
	<ol>
		<li>Prepare the appropriate media for either hippocampal neurons19 or dorsal root ganglion neurons24, which is to be warmed in a 37 &#176;C water bath prior to use. Incubate the dishes overnight in the CO2 incubator.</li>
		<li>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 &#176;C water bath prior to use.</li>
		<li>For DRG neurons, the day following dissection/plating, prepare a fresh stock of media with an anti-mitotic agent (Uridine and 5&#39;-Fluoro-2&#39;-deoxyuridine)&#160;make a 10 mM stock solution of each, separately; use a final concentration of 10 &#181;M for each. Remove half of the DRG media (875 &#181;l) and add 875 &#181;l of fresh media with the anti-mitotic agent.
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Vitrifying Neurons on EM Grids
<ol>
	<li>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.</li>
	<li>Start the vitrification device. Set the humidity to 100% and temperature to 32 &#176;C. In the &#8220;Console&#8221; section, set the blot time to zero seconds. This allows for manual blotting through the side-window of the vitrification machine&#39;s humidity chamber.</li>
	<li>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&#176; angle such that one side of the paper has a 0.5 cm x 0.5 cm face (Figure&#160;2). Using tweezers, remove the middle paper and place it on another calcium-free filter paper until use.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;s flushed with the bottom of the humidity chamber.</li>
	<li>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&#160;2B). Carefully insert the filter paper into the side-hole of the vitrification machine&#39;s humidity chamber (Figure&#160;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&#39;s automation.</li>
	<li>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.</li>
</ol>
4. Image Collection, Processing, and Annotation
<ol>
	<li>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.
	<ol>
		<li>In this case, all images were collected using a 4k x 4k CCD camera attached to a 200&#160;kV electron microscope equipped with a single tilt liquid nitrogen cryo transfer holder, at 20k&#160;microscope magnification at a target underfocus of 7 &#181;m and sampling of 4.4 &#197;/pixel.</li>
		<li>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&#176; to 60&#176; at 5&#176; increments with a cumulative dose of ~60 e/&#197;2.</li>
	</ol>
	</li>
	<li>Reconstruct the tilt series of the neurites using image processing software21 as previously described for other samples5,29.</li>
	<li>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.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental neuroscience. , (2003).</li><li>Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+hit+hypotheses+for+dopamine+neuron+loss+in+Parkinson's+disease.">Multiple hit hypotheses for dopamine neuron loss in Parkinson's disease.</a> Trends Neurosci. 30, 244-250 (2007).</li><li>Tapia, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+expression+of+glycine+and+GABA(A)+receptors+in+developing+spinal+cord+neurons.+Effects+on+neurite+outgrowth.">Early expression of glycine and GABA(A) receptors in developing spinal cord neurons. Effects on neurite outgrowth.</a> Neuroscience. 108, 493-506 (2001).</li></ol>

The authors declare that they have no competing financial interests.

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

79.1K Views.  Baylor College of Medicine.  כדי לשמר את התהליכים עצביים לניתוח ultrastructural, אנו מתארים פרוטוקול לציפוי של נוירונים העיקרי ברשתות במיקרוסקופ אלקטרונים ואחרי הבזק הקפאה, מניב דגימות טהורים בשכבה של קרח זגוגי. ניתן לבחון דגימות אלה עם מיקרוסקופ אלקטרונים cryo-לדמיין מבנים ב...

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

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Measuring Connectivity in the Primary Visual Pathway in Human Albinism Using Diffusion Tensor Imaging and Tractography

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed as candidate sites for misrouting. Since a correlation between the number of lateral geniculate nucleus (LGN) relay neurons and LGN size has been shown, and based on previously reported reductions in LGN volumes in human albinism, we suggest that fiber projections from LGN to the primary visual cortex (V1) are also reduced. Studying structural differences in the visual system of albinism can improve the understanding of the mechanism of misrouting and subsequent clinical applications. Diffusion data and tractography are useful for mapping the OR (optic radiation). This manuscript describes two algorithms for OR reconstruction in order to compare brain connectivity in albinism and controls.An MRI scanner with a 32-channel head coil was used to acquire structural scans. A T1-weighted 3D-MPRAGE sequence with 1 mm3 isotropic voxel size was used to generate high-resolution images for V1 segmentation. Multiple proton density (PD) weighted images were acquired coronally for right and left LGN localization. Diffusion tensor imaging (DTI) scans were acquired with 64 diffusion directions. Both deterministic and probabilistic tracking methods were run and compared, with LGN as the seed mask and V1 as the target mask. Though DTI provides relatively poor spatial resolution, and accurate delineation of OR may be challenging due to its low fiber density, tractography has been shown to be advantageous both in research and clinically. Tract based spatial statistics (TBSS) revealed areas of significantly reduced white matter integrity within the OR in patients with albinism compared to controls. Pairwise comparisons revealed a significant reduction in LGN to V1 connectivity in albinism compared to controls. Comparing both tracking algorithms revealed common findings, strengthening the reliability of the technique.

This manuscript describes deterministic and probabilistic algorithms for white matter (WM) reconstruction, used to examine differences in optic radiation (OR) connectivity between albinism and controls. Although probabilistic tractography follows the true course of nerve fibers more closely, deterministic tractography was run to compare the reliability and reproducibility of both techniques.

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...