Name: assessment of ultrastructural neuroplasticity parameters after in utero transduction of the developing mouse brain and spinal cord
Uploaded: 2019-02-26T14:45:00
Description: Watch this Scientific Journal Video about Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord at JoVE.com

The authors thank the colleagues of the animal facility at the Medical Faculty, Ruhr-University Bochum, for their support and animal care.

All procedures on animal subjects have been approved by the institutional animal ethics committees of the federal states of Hamburg and Nordrhein-Westfalen, Germany.&#160;&#160;Use sterile instruments, protective gloves and aseptic coats throughout the entire surgical procedure.
1. In Utero Transduction
<ol>
	<li>Prepare adeno-associated virus type 1 (AAV1) coding for the desired target (4 x 1011 viral particles/&#181;L of AAV1) in phosphate-buffered saline (PBS) at pH 7.4. Add 0....

<ol>
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H., 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=Presenilins+regulate+synaptic+plasticity+and+mitochondrial+calcium+homeostasis+in+the+hippocampal+mossy+fiber+pathway.">Presenilins regulate synaptic plasticity and mitochondrial calcium homeostasis in the hippocampal mossy fiber pathway.</a> Molecular Neurodegeneration. 12, 48 (2017).</li><li>LoTurco, J., Manent, J. 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A crucial step of in utero transduction is the injection procedure. The precise injection into brain ventricles or into another area of interest requires experience and hands-on skill. The thinner the microcapillary tip, the less tissue damage may occur; however, this is at the cost of increasing injection pressure. In contrast to in utero electroporation19,20,21,22, the survival rate of the in...

No photon can penetrate an ultrathin tissue specimen in the depth grade of an electron. This attributes invaluable advantages to TEM in capturing nanometer resolution images of fine structures when compared to light microscopy techniques. For example, TEM allows for the visualization of intracellular organelles such as mitochondria, melanosomes, and various types of secretory granules, microtubules, microfilaments, cilia, microvilli, and intercellular junctions (cell surface specializations), in particular synapses in the nervous system1,2,3,4. The overall goal of the present methodological study is the ultrastructural recognition of changes in neural plasticity during development upon prenatal interference by combining the state-of-the-art techniques of in utero transduction and TEM. Virally encoded proteins of interest have been transduced in utero into the central nervous system5,6,7, including the spinal cord6. For instance, in utero transduction in combination with TEM has been used for studying the effect of the cell adhesion molecule L1 on motor learning plasticity in L1-deficient mice, in particular with regard to the interplay between L1 and nuclear receptor proteins in cerebellar neurons7.
The analysis of neuroplasticity parameters requires precise information about the localization of the smallest areas within the nervous system. Therefore, it is adequate to describe ultrastructural details and their exact topographical orientation with respect to other structures. In the present study, a specific preparatory method aiming at the detailed investigation of distinct morphological areas based on both light and electron microscopy is presented. This approach combines several techniques of tissue manipulation, starting with in utero transduction of the mouse brain and spinal cord and followed by perfusion fixation, mold-embedding, and processing the tissue for TEM. An essential step included between the embedding and the processing of the tissue for TEM is the documentation of the tissue, using the interference light reflection technique that allows for the precise microphotographic and low-magnification documentation of tissue specimens8,9,10. Incorporated into the present approach, this technique enables researchers to examine topographical and structural details of nervous tissue surfaces and of specimen slice profiles prior to their preparation for TEM.
A special frame for sectioning whole brains corresponds to stereotaxic coordinates. This frame benefits the morphological three-dimensional (3D) reconstruction of areas in nervous tissue and can be used for morphometric analysis. The macrographs of the visualized sections are assigned topographical coordinates, and the serially numbered sections build maps in a tissue atlas.
After resin processing, the embedded tissue is sectioned into ultrathin sections (&#60;70 nm) containing selected areas, according to the maps of the above-mentioned tissue atlas. The ultrathin sections are subjected to TEM to obtain high-resolution images of plasticity parameters (e.g., cross-section profile areas of synaptic boutons or axonal fibers) of their contents and of contacts to neighboring structures within the complex neuropil.
With the method described herein, the smooth transition from visualized macrostructures to micro- and nanostructures permits comparative in-depth studies of morphological neuronal plasticity after in utero transduction of the developing nervous system.

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			<td height="40" style="height:40px;width:257px;">2,4,6-Tris(dimethyl-aminomethyl)phenol</td>
			<td style="width:167px;">Serva</td>
			<td style="width:123px;">36975</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">26Gx 1&#39;&#39; needle</td>
			<td style="width:167px;">Henke-Sass, Wolf GmbH</td>
			<td style="width:123px;"></td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td>410 Anaesthesia Unit for air pump</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">8323102</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="414">
			<td>Adeno-associated virus serotype 1 (AAV1)</td>
			<td>UKE (Viral Core Facility)</td>
			<td>-</td>
			<td>For references and target areas of AAV1 see: https://www.addgene.org/viral-vectors/aav/aav-guide/ and also: Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Kwon I, Schaffer DV. Pharm Res. 2008 Mar;25(3):489-99. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N. Mol. Ther. 2004 Aug;10(2):302-17. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54.</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Agarose</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:123px;">A9414</td>
			<td style="width:423px;">low gelling agarose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Air Pump</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">Eheim 100</td>
			<td style="width:423px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Araldite</td>
			<td style="width:167px;">CIBA-GEIGY</td>
			<td style="width:123px;">23857.9</td>
			<td style="width:423px;">resin for embedding of tissue</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">aspirator tune assemblies</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:123px;">A5177-5EA</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Breathing Mask Mouse Anodized Aluminium</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">buprenorphine</td>
			<td style="width:167px;">Temgesic</td>
			<td style="width:123px;">ampules</td>
			<td style="width:423px;">painkiller</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">capillaries</td>
			<td style="width:167px;">Science-Products</td>
			<td style="width:123px;">GB100TF-10</td>
			<td style="width:423px;">with fillament</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Dodecenylsuccinic anhydride</td>
			<td style="width:167px;">Fluka</td>
			<td style="width:123px;">44160</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Dumont tweezers (#3, 12 cm, straight, 0.2 x 0.12 mm)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">11203-23</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">electric shaver</td>
			<td style="width:167px;">Phillips</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Ethicon sutures (Ethilon, 6-0 and 3-0)</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">polyamide</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">eye lubricant</td>
			<td style="width:167px;">Bepanthene</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
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		<tr height="52">
			<td height="52" style="height:52px;width:257px;">Fast Green</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:123px;">F7252</td>
			<td style="width:423px;">for visualization of injected liquids</td>
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		<tr height="133">
			<td height="133" style="height:133px;width:257px;">Gas Routing Switch 4/2 connectors</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">8433020</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;width:257px;">halsted Mosquito hemostatic forceps (12.5 cm, straight)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">13011-12</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:257px;">Heparin-Natrium</td>
			<td style="width:167px;">Ratiopharm</td>
			<td style="width:123px;"></td>
			<td style="width:423px;">25 000 I.E./5 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Induction box for mice 
			with horizontally moving lid. 
			Inner dimensions: LxBxH: 155x115x130 mm. 
			Wall thickness: 6 mm</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">iris forceps (10cm, curved, serrated)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">14007-14</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">iris scissors (11cm, straight, tungsten carbide)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">14501-14</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Isofluran OP Tisch, electrically heated, sm 
			Outer dimensions: 257x110x18 mm. 
			Heating area: 190x90 mm 
			The removal of the isoflurane escaping 
			the breathing mask is downwards in compliance with the 
			regulations</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">isoflurane (Attane)</td>
			<td style="width:167px;">JD medical</td>
			<td style="width:123px;"></td>
			<td style="width:423px;">inhalation anesthesia</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">LED RGB lights</td>
			<td style="width:167px;">Cameo</td>
			<td style="width:123px;">CLQS15RGBW</td>
			<td style="width:423px;">LEDs 2 x 15 W</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Light microscope Basic DM E</td>
			<td style="width:167px;">Leica</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">4x (N.A. 0.1 &#8734;/-), 10x (N.A. 0.22 &#8734;/0.17), 40x (N.A. 0.65 &#8734;/0.17), 100x (N.A. 1.25 &#8734;/0.17) objectives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">micropipette puller</td>
			<td style="width:167px;">Science-Products</td>
			<td style="width:123px;">P-97</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Mosquito hemostatic forceps (12.5cm, curved)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">13010-12</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Nickel grids, 200 mesh</td>
			<td style="width:167px;">Ted Pella</td>
			<td style="width:123px;">1GC200</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Osmium (VIII)-oxid</td>
			<td style="width:167px;">Degussa</td>
			<td style="width:123px;">73219</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Propylene oxide</td>
			<td style="width:167px;">Fluka</td>
			<td style="width:123px;">82320</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">razor blades</td>
			<td style="width:167px;">Schick</td>
			<td style="width:123px;">87-10489</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Sodium pentobarbital (Narcoren)</td>
			<td style="width:167px;">Merial GmbH</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TC01mR 1-Channal temperature controller with feedback</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Technovit 4004 two components glue</td>
			<td style="width:167px;">Kulzer</td>
			<td style="width:123px;"></td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:257px;">Telemacrodevice</td>
			<td style="width:167px;">Canon</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">Canon Spiegelreflex Kamera EOS2000D, EF-S 18-55 mm f/3.5-5.6 IS STM Objective, Extension below 150 mm, Manual Extension Tube 7 mm ring, 14 mm ring, 28 mm ring, Macro reverse ring (58 mm), Canon copy stand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Thermopuller P-97</td>
			<td style="width:167px;">Sutter Instruments</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">thin vibrating razor blade device</td>
			<td style="width:167px;">Krup</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">with Szabo thin blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">toluidine blue</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td align="right">89640</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Transmission electron microscope C20</td>
			<td style="width:167px;">Phillips</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">up to 200 kV</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:257px;">Tygon 6/4 Tubing material for connection of all parts 
			Outer diameter: 6mm 
			Inner diameter: 4mm 
			Wa 
			ll thickness: 1mm</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Ultracut E</td>
			<td style="width:167px;">Reichert-Jung</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">ultramicrotome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Univentor Scavenger</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">8338001</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Vannas scissors (8 cm, straight)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">15009-08</td>
			<td style="width:423px;"></td>
		</tr>
	</tbody>
</table>

assessment of ultrastructural neuroplasticity parameters after in utero transduction of the developing mouse brain and spinal cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

For reliable and fast anesthesia of mice, numerous safety parameters were considered, and an optimized workspace of the anesthesia unit proved to be adequate (Figure 1A). The unit is designed to control the mixture of liquid isoflurane and ambient air with a precision required for successful surgery on small animals, such as mice and rats. Air and isoflurane are mixed in the vaporizer according to the desired settings and delivered into a box...

Watch this Scientific Journal Video about Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord at JoVE.com

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Our combined approach helps answer how ultrastructural neuroplasticity parameters change after early developmental interventions to the nascent nervous system in utero. The main advantage of our method is the acquisition of high-resolution images of macro-meso-micro-and nanostructures in the predefined coordinate system within a tissue atlas. The implication of our method is its translational therapeutic potential for congenital neurodegenerative diseases.For example, treatment and rescue at embryonic stages using this in utero transduction technique. Our method can be applied to any other model. For example, the mouse species can be changed, as well as the area of interest.Researchers trying our method for the first time need profound surgical training, as well as the knowledge of how to prepare the tissue for electron microscopic imaging. Begin by loading a customized capillary tip containing four times 10 to the 11 viral particles of adeno-associated virus type 1 coating for the desired target, and labeled with Fast Green dye onto an aspirator tube. Add 15 microliters of the labeled adeno-associated virus particles into the capillary.Next, confirm a lack of response to toe pinch in an anesthetized embryonic day 14.5 pregnant mouse, and disinfect the skin. Use curved serrated iris forceps and straight tungsten carbide scissors to make a skin incision along the linea mediana. Using straight Dumont tweezers to grip the peritoneal wall, continue along the linea alba with straight Vannas scissors and place a piece of fenestrated paraffin film over the abdominal opening and use a spoon-like device to expose the uterine horns without damaging the embryos inside the horns.After applying a few drops of 37-degree-Celsius PBS onto the uterine horns, inspect the embryos for damage or malformations inside the uterine sac. Turn the embryos carefully inside their sacs until the desired position for each injection is achieved. When the embryos are in place, inject one to two microliters of the Fast Green labeled virus particle solution into each embryo.After all of the embryos have been injected, place the uterine horns back into the abdominal cavity, and add a few drops of 37-degree-Celsius PBS into the abdomen. Then use polyamide 6-0 sized sutures to close the peritoneal wall and polyamide 3-0 sized sutures and Halsted's Mosquito hemostatic forceps to close the skin. To embed isolated tissues of interest for tele-macro photography, place the tissue sample into a special frame with the reproducible sectioning angle, and document the coordinates.Fill the frame with 30-degree-Celsius 3%agarose until the tissue is covered, and cover the frame with a plastic lid until the agarose has hardened. While the agarose is hardening, use the tele-macro graphic device to image the embedded tissue and its coordinates within the frame. When the agarose has solidified, transfer the agarose-embedded tissue into the frame with cutting gaps corresponding to the coordinates of the first frame.Use a device with a thin and vibrating razor blade to cut the embedded tissue into sections of an appropriate experimental thickness. Then image each tissue section in PBS, and collect the images into a folder. To prepare the tissue samples for transmission electron microscopy, in a biosafety cabinet, wash the tissue sections with two 30-minute washes in PBS, followed by a two-hour incubation in 2%aqueous osmium tetroxide solution at room temperature.At the end of the incubation, wash the osmicated sections two more times in PBS for 30 minutes per wash and dehydrate the sections in sequential 10-to 15-minute ethanol immersions as indicated. After the 70%ethanol immersion, place each sample into a black dish on a dull black background and image the specimens in 70%ethanol under LED RGB light applied to the samples from the left and right sides at 45-degree angles. Create an atlas of the section images with coordinates by collecting images of the samples in series in a folder.Treat the specimens with two 30-minute 100%ethanol incubations, followed by two 30-minute 100%propylene oxide incubations, both at room temperature. While the samples are incubating, mix freshly prepared resin with propylene oxide at one-to-one and one-to-two ratios, and add 3%accelerator to both solutions. After the second propylene oxide incubation, transfer the samples into the one-to-two resin propylene oxide embedding medium solution for two hours at room temperature, followed by a two-hour incubation in the one-to-one resin propylene oxide embedding medium solution at room temperature.At the end of the second incubation, transfer the tissues into flat polypropylene dishes, and cure the embedded tissues with fresh resin containing 3%accelerator for 12 to 24 hours at 65 to 85 degrees Celsius. Then cool the tissue to room temperature, and remove the resin-embedded specimens from the polypropylene dishes. To map an area of interest, select an image containing the area of interest from the previously prepared image atlas.Sketch the borders of the area of interest onto the sectioned image, optically superimpose these borders onto the embedded specimen under the microscope, and use a one-inch 26 gauge needle to scratch these region borders onto the resin specimen. Then, heat the specimens to 95 degrees Celsius in order to soften the resin. Next, take out the warm resin specimens from the oven, and use a razor blade to excise the areas of interest.Glue the specimens onto acrylic glass holding bars of the appropriate caliber, and trim the mounted specimens for semi-and ultra-thin sectioning. Use an ultramicrotome to acquire semi-thin 0.7-micrometer and ultra-thin 70-nanometer sections, collecting the semi-thin sections on glass carriers, and the ultra-thin sections on nickel grids. Stain the semi-thin sections with 1%Toluidine blue in PBS for four minutes, followed by several washes in deionized water, before examining the sections by light microscopy.The ultra-thin sections can then be directly assessed by transmission electron microscopy at 180 kilovolts without further modification. After sectioning, the tissue slices are imaged by tele-macro photography as just demonstrated. After osmication and ethanol immersion, the sections are imaged again by interference light tele-macrography, revealing a specifically colored pattern for each tissue surface.After additional dehydration and resin embedding, the areas of interest are selected and further processed for transmission electron microscopy. In the hippocampus and cerebellum, mossy fiber synapses are known to exhibit unique ultrastructural characteristics when compared to other synapses, including large presynaptic boutons. In their cross-section profiles, the boutons contain a vast number of vesicles and mitochondria, and very often enclose several dendritic spines.In the spinal cord, the dorsal funiculus contains many axonal fibers that are myelinated by oligodendroglia. On transversal sections, the dorsal funiculus can be found between both posterior horns of the spinal cord. On transmission electron microscopy images, cross-sectioned myelinated axons appear round, and the myelin sheaths surrounding the axons are organized in the lamellae.The prepared atlas and the micrographic transmission electron microscopy images of the ultrastructural parameters are not only helpful for comparison of the morphological neuroplasticity of different sections under various treatment conditions, but also for comparisons between parameters within the same area. Remember to thoroughly plan and prepare before starting the in utero transduction, and to be sure to have backup of materials and instruments in case of complications. Following our method, other techniques can be added such as immunogold labeling prior to electron microscopy for ultrastructural tracing of a defined molecule of interest.Starting with a holistic view of the entire organism and zooming in towards the molecular topography, we are able to study and manipulate the relationship between tissue function and morphology. Osmium tetroxide is hazardous. Be sure to always work under the hood, and to wear protective glasses and hand-gloves when working with this reagent.

Research

JoVE Journal

Developmental Biology

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