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 0.1 mg/&#181;L Fast Green and keep the AAV1-Fast-Green mixture at 37 &#176;C.</li>
	<li>Prepare a thin capillary tip with the desired shape (8 mm in length, with an outside diameter of 80 &#181;m and an inside diameter of 50 &#181;m), using a micropipette puller (settings: pressure = 500, heat = 700, pull = 0, velocity = 80, time = 200, see the Table of Materials). Break the tip of the capillary so that it is 4-5 mm.</li>
	<li>Assemble an aspirator tube (44 cm x 0.7 cm) with the capillary tip and aspirate 15 &#181;L of the AAV1-Fast-Green mixture into the capillary.</li>
	<li>Keep the animal subjects at a constant physiological body temperature of 37 &#176;C throughout the entire procedure.</li>
	<li>Place a pregnant C57Bl/6 mouse (embryonic day 14.5) into the preincubation chamber and anesthetize the mouse with gaseous 4% isoflurane (with a volumetric airflow rate of 0.6-0.8 L/min).</li>
	<li>Subcutaneously, inject buprenorphine (0.1 mg/kg of body weight).</li>
	<li>Place the anesthetized mouse on the prewarmed surgical plate (37 &#176;C).</li>
	<li>Cover the eyes with a lubricant.</li>
	<li>Fit the mouse with the anesthesia mask (gaseous 1.5 % isoflurane at a volumetric airflow rate of 0.6-0.8 L/min)on the surgical plate and shave the abdominal skin region.&#160;Wipe the shaved region with 3X 75% ethanol and then with betadine solution. 
	NOTE: Monitor the breathing behavior of the anesthetized mouse continuously. Adjust the concentration of the isoflurane gas according to the inhalation-exhalation pattern of the mouse.</li>
	<li>Check for the absence of the plantar reflex by squeezing the hind paw phalanges of the mouse.</li>
	<li>Open the abdominal cavity by gripping the skin with curved serrated iris forceps (10 cm) and cutting the skin along the linea mediana with straight tungsten carbide scissors (10 cm), and then, by gripping the peritoneal wall with straight Dumont tweezers (12 cm, 0.2 mm x 0.12 mm) and cutting the wall along the linea alba with straight Vanna&#39;s scissors (8 cm).</li>
	<li>Place a piece of fenestrated paraffin film on the abdominal opening and fix the film on both ends with micro-mosquito hemostatic forceps (12.5 cm, curved).</li>
	<li>Expose the uterine horns with a spoon-like device to avoid damage to the embryos inside the uterine horns. Drip a few drops of PBS (37 &#176;C) on the uterine horns and inspect the embryos for damages or malformations inside the uterine sac.</li>
	<li>Document the order and position of the embryos in the uterine horns. Turn the embryos carefully inside the uterine sac until the desired position for injection is reached.</li>
	<li>Inject 1-2 &#181;L of the AAV1-Fast-Green mixture by visually inspecting the injection site (e.g., brain ventricles) and the dye penetration under a stereomicroscope.</li>
	<li>Document the injected embryos and place the uterine horns with the injected embryos back into the abdominal cavity.</li>
	<li>Drip a few drops of PBS (37 &#176;C) into the abdominal cavity. Close the cavity by suturing the peritoneal wall (use polyamide 6-0-sized sutures) and the skin (use polyamide 3-0-sized sutures), using Halsted&#39;s mosquito hemostatic forceps (12.5 cm, curved).&#160;Alternatively, use a simple interrupted pattern or stainless-steel suture/staples to close the abdominal cavity and the skin.</li>
</ol>
2. Telemacrophotography of Isolated Tissues
<ol>
	<li>Preparation of buffers
	<ol>
		<li>Prepare S&#246;rensen&#39;s buffer (1 L) by dissolving 14.95 g of Na2HPO4 and 2.18 g of KH2PO4 in 1 L of distilled water under stirring at 200 rpm.&#160;For perfusion of late gestation pups and/or pups in the post-natal time periods, use an appropriate size of needles for abdominal or intragastric injection and make sure that the concentration of the terminal sodium pentobarbital anesthesia does not exceed 180 mg/kg of body weigh.</li>
		<li>Prepare Mugnaini&#39;s fixation solution (5 L) by heating 500 mL of distilled water to 75 &#176;C and adding 50 g of paraformaldehyde powder under stirring at 200 rpm, adding 200 &#181;L of 5 N NaOH, adding 1,500 mL of S&#246;rensen&#39;s buffer, 1,750 mL of distilled water, and 500 mL of 25% glutaraldehyde. Fill up to 5,000 mL with distilled water. Use this final buffer for perfusion. 
		NOTE: Prepare Mugnaini&#39;s fixation solution under the hood, wear protective glasses, and avoid fumes. Add methylene blue (0.05 g/L) for a better visualization of the perfusion.</li>
	</ol>
	</li>
	<li>Mouse perfusion and tissue isolation
	<ol>
		<li>Transcardially perfuse the pregnant mice that carry the transduced embryos (in the case of embryo studies) or the born transduced pubs at the desired age (e.g., postnatal day 24) according to standard procedures6,7,11,12,13,14,15,16,17, using intraperitoneal terminal sodium pentobarbital anesthesia (200 mg/kg of body weight).</li>
		<li>Inject the mice transcardially with heparin solution (500 U) using a 26 G, 1 in needle and, before fixation, infuse the mice transcardially with 10 mL of PBS to flush out the blood from the body, and perfuse them transcardially with 30 mL of 40 &#176;C prewarmed Mugnaini&#39;s fixation solution. 
		NOTE: For adult mice, perform an alternative retrograde perfusion via the abdominal aorta14.</li>
		<li>Isolate the perfused tissue of interest (e.g., whole brain or spinal cord) and postfix the tissue in at least 10 mL of Mugnaini&#39;s fixation solution for another 24 h at 4 &#176;C.</li>
		<li>Wash the tissue in 10 mL of PBS for 3 h at room temperature.</li>
	</ol>
	</li>
	<li>Embedding in agarose, plus documentation and sectioning
	<ol>
		<li>Adjust the isolated tissue (e.g., whole brain) in a special frame with a reproducible sectioning angle8,9,10. Alternatively, use a vibratome with an adjustable cutting thickness.</li>
		<li>Place the nervous tissue in the frame, adjust the tissue for telemacrography, and document the coordinates.</li>
		<li>Prepare 3% low-melting agarose-embedding medium: add 3 g of agarose in 100 mL of S&#246;rensen&#39;s buffer and heat the mixture in a water bath to 90 &#176;C.</li>
		<li>Pour 3% agarose (30 &#176;C) in the frame that contains the tissue. Cover the frame with a warm metal block and wait until it is hardening. During hardening, use telemacrographic devices to image the embedded tissue and its coordinates within the frame.</li>
		<li>Transfer the agarose-embedded tissue into a frame with cutting gaps corresponding to the coordinates of the first frame.</li>
		<li>Cut the embedded tissue into sections of desired thickness (e.g., 1.5 mm) with a device with a thin and vibrating razor blade (see Table of Materials). 
		NOTE: To improve the gliding of the razor blade, drip a few drops of glycerin onto the embedded tissue.</li>
		<li>Image each tissue section in PBS and collect the images into a folder.</li>
	</ol>
	</li>
</ol>
3. Preparation of the Isolated Tissue for Transmission Electron Microscopy
NOTE: Perform all further steps of incubation in glass dishes with tightly closable lids on a shaking platform under the hood.
<ol>
	<li>Wash the tissue sections for 2x 30 min in PBS. Incubate the sections in 2% aqueous osmium tetroxide solution (OsO4) for 2 h at room temperature. 
	CAUTION: Osmium tetroxide is toxic and may be harmful when it comes in contact with skin.</li>
	<li>Wash the osmicated sections for 2x 30 min in PBS.</li>
	<li>Incubate the sections in 30%, 50%, and 70% ethanol at room temperature for 10-15 min (optional: incubate in 70% ethanol at 4 &#176;C overnight).</li>
	<li>Image the osmicated specimens in 70% ethanol under LED RGB light8,9,10 (2x 15 W) applied to the sample from the left and right side at an angle of 45&#176;. Use black dishes and a dull black background to minimize scattering and the reflection of the light during illumination. 
	CAUTION: Do not allow the section to dry out during imaging.</li>
	<li>Create an atlas of the section images with coordinates by collecting images in series in a folder.</li>
	<li>Incubate the specimens in 100% ethanol (2x for 30 min) and 100% propylene oxide (2x for 30 min) at room temperature. 
	CAUTION: Do not allow the sections to dry out while changing solutions.</li>
	<li>Mix 260 mL of resin with 240 mL of dodecenylsuccinic anhydride in a glass vessel while gently stirring with a glass bar. Periodically check for inhomogeneity, bubbles, and smears. Very gently, stir by hand for at least 45 min.</li>
	<li>Prepare resin/propylene oxide in a ratio of 1:2 and 1:1 and add 3% accelerator (2,4,6-Tris(dimethylaminomethyl)phenol).</li>
	<li>Incubate the tissue in the 1:2 embedding solution from step 3.8 for 2 h and then in the 1:1 embedding solution from step 3.8 for 2 h at room temperature on a rotating wheel.</li>
	<li>Place the tissue in flat polypropylene dishes, cover the tissue with fresh resin containing 3% accelerator, and cure the embedded tissue at 65-85 &#176;C for 12-24 h.</li>
	<li>Cool down the embedded tissue to room temperature and remove the resin-embedded specimens from the polypropylene dishes.</li>
</ol>
4. Selection of Ultrastructural Neuroplasticity Parameters for Quantitative Analysis
<ol>
	<li>Mapping the area of interest
	<ol>
		<li>Choose an area of interest (e.g., hippocampus or cerebellum) and localize the area in the section atlas by choosing the image from the atlas (step 3.5) that contains this area.</li>
		<li>Sketch the borders of the area of interest onto the section image and find/superimpose these region borders onto the resin specimen.</li>
		<li>Scratch-mark the borders of the area of interest (e.g., hippocampus or cerebellum) on the resin specimen, using a fine needle gauge (26 G, 1 in).</li>
		<li>Heat the resin specimen to 85 &#176;C in an oven to soften the resin for trimming or, alternatively, use a trimming device, a thin blade, or sandpaper.</li>
		<li>Excise the area of interest from the resin specimen with a razor blade (see Table of Materials). Mount the specimen on holding bars of acrylic glass of the required caliber (e.g., with a diameter of 8 mm and a length of 1 cm) with glue. Trim the mounted specimen for semi- and ultrathin sectioning.</li>
		<li>Prepare semithin (0.75 &#181;m) and ultrathin (70 nm) sections of the trimmed area using an ultramicrotome: set it at 1.5 mm/s for 0.75 &#181;m thickness and at 0.7 mm/s for 70 nm thickness.</li>
		<li>Collect the semithin sections on glass carriers and stain the sections with 1% toluidine blue in PBS (for 4 min).</li>
		<li>Wash the sections several times in deionized water. Examine the stained sections under the light microscope using 4x (NA of 0.1 &#8734;/-), 10x (NA of 0.22 &#8734;/0.17), 40x (NA of 0.65 &#8734;/0.17), and 100x (NA of 1.25 &#8734;/0.17) objectives.</li>
		<li>Collect ultrathin sections on nickel grids. Subject the grids to TEM at 180 kV and at 3,200x, 6,000x, and/or 8,000x magnification.</li>
	</ol>
	</li>
	<li>TEM Analysis
	<ol>
		<li>Choose the ultrastructural parameters of interest for quantitative TEM analysis (e.g., boutons with vesicles and mitochondria or myelinated and nonmyelinated axons) and take TEM images of these parameters under 3,500x, 6,000x and/or 8,000x magnification.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:969px;" width="968">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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 ...

assessment-ultrastructural-neuroplasticity-parameters-after-utero

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.8K Views.  Ruhr University Bochum. 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.

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

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

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Chapters in this video

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

Technique to Target Microinjection to the Developing Xenopus Kidney

Live Imaging of Mouse Secondary Palate Fusion

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina