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 injected embryos after in utero transduction is very high. All embryos of the uterine horn can be injected, even if embryos are located at the roots of the uterine horns and need to be readjusted within the uterine sac. The developmental stages of injection and analysis can be adapted to the scientific question.
When injected into the brain ventricles, the AAV1 particles are distributed via the cerebrospinal fluid through the entire ventricular system. Developing structures with surfaces that are in close contact with the liquor, such as the hippocampus and the cerebellum, are transduced by the applied viral particles5,6,7. An analysis of the changes in ultrastructural parameters, such as bouton size, cross-sectioned bouton areas, numbers of synaptic vesicles, numbers of mitochondria, and numbers and length of synaptic contacts in these structures, is an elegant read-out for ultrastructural plasticity. The in utero transduction method can be applied to study different brain areas, as well as the spinal cord6. Moreover, other organs and tissues can similarly be chosen as transduction targets. Finally, the frequently used mouse model can be replaced by other species as required.
The photography of the tissue slices allows for a very precise dissection of resin-embedded samples for transmission electron microscopy. Since the spatial orientation of the sample is defined, extensive and time-consuming cutting was omitted. Moreover, crucial neighboring regions can be kept in their original relationship to each other. An important advantage of the photographic documentation step is the possibility to monitor the quality of tissue fixation and to recognize pathological alterations. Thus, the quality of fixation and embedding of the material is of pivotal importance. Nonsuitable samples that contain artifacts can be outsourced prior to further processing.
The micrograph series of the imaged tissue slices provide a helpful morphological atlas. For comparative neuroanatomical studies, which may also include rare species, the atlas is a valuable archive. The choice of thickness of the tissue slices depends on the addressed scientific question and is limited by the size of the tissue specimen. For small animals, a thickness of up to 1 mm should be adjusted. Very thick sections (&#62;5 mm) are not recommended, due to the limited penetration capacity of osmium tetroxide8,9,10.
The telemacrophotography documentation is essential for morphometrical studies. It offers a fixed correlation of the tissue surface to the side and top plane of the frame and defines the position of a given structure in a spatial system of coordinates. A combination of the described method with electrophysiology18 or with the spinal cord injury model, where plasticity plays an essential role in regeneration6, is possible. For comparative neuroanatomical studies, which may also include rare species, the iridescent tissue atlas and the micrographic TEM images of the ultrastructural parameters are valuable assets.
Despite its strengths, TEM has several limitations due to the thickness size of the sample (70 nm), the cross-sectioned area (&#60;6 mm2), and the complexity of the sample preparation. However, when a specimen is accurately prepared, the TEM images of the specimen&#39;s ultrastructure are of superior quality in comparison to images produced by any light-microscopical techniques. The in utero transduction technique is limited by the injected volume and the topographical region of the injection. Indisputably, the technique has a strong translational medical potential in early developmental treatment and in the rescue of congenital neuropathological diseases. Moreover, the present combination of in utero transduction with TEM might not only be considered as an approach for the future treatment of congenital diseases, but also for high-quality ultrastructure-based neurodevelopmental diagnostics.

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><tbody><tr><td>2,4,6-Tris(dimethyl-aminomethyl)phenol</td><td>Serva</td><td>36975</td><td></td></tr><tr><td>26 G x 1&#039;&#039; needle</td><td>Henke-Sass, Wolf GmbH</td><td></td><td></td></tr><tr><td>410 Anaesthesia Unit for air pump</td><td>Biomedical Instruments (Univentor)</td><td>8323102</td><td></td></tr><tr><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><td>Agarose</td><td>Sigma-Aldrich</td><td>A9414</td><td>low gelling agarose</td></tr><tr><td>Air Pump</td><td>Biomedical Instruments (Univentor)</td><td>Eheim 100</td><td></td></tr><tr><td>Araldite</td><td>CIBA-GEIGY</td><td>23857.9</td><td>resin for embedding of tissue</td></tr><tr><td>aspirator tune assemblies</td><td>Sigma-Aldrich</td><td>A5177-5EA</td><td></td></tr><tr><td>Breathing Mask Mouse Anodized Aluminium</td><td>Biomedical Instruments (Univentor)</td><td>-</td><td></td></tr><tr><td>buprenorphine</td><td>Temgesic</td><td>ampules</td><td>painkiller</td></tr><tr><td>capillaries</td><td>Science-Products</td><td>GB100TF-10</td><td>with fillament</td></tr><tr><td>Dodecenylsuccinic anhydride</td><td>Fluka</td><td>44160</td><td></td></tr><tr><td>Dumont tweezers (#3, 12 cm, straight, 0.2 x 0.12 mm)</td><td>FST</td><td>11203-23</td><td></td></tr><tr><td>electric shaver</td><td>Phillips</td><td>-</td><td></td></tr><tr><td>Ethicon sutures (Ethilon, 6-0 and 3-0)</td><td>Ethicon</td><td>-</td><td>polyamide</td></tr><tr><td>eye lubricant</td><td>Bepanthene</td><td>-</td><td></td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7252</td><td>for visualization of injected liquids</td></tr><tr><td>Gas Routing Switch 4/2 connectors</td><td>Biomedical Instruments (Univentor)</td><td>8433020</td><td></td></tr><tr><td>halsted Mosquito hemostatic forceps (12.5 cm, straight)</td><td>FST</td><td>13011-12</td><td></td></tr><tr><td>Heparin-Natrium</td><td>Ratiopharm</td><td></td><td>25 000 I.E./5 mL</td></tr><tr><td>Induction box for mice&lt;br/&gt; with horizontally moving lid.&lt;br/&gt; Inner dimensions: LxBxH: 155 mm x 115 mm x 130 mm.&lt;br/&gt; Wall thickness: 6 mm</td><td>Biomedical Instruments (Univentor)</td><td>-</td><td></td></tr><tr><td>iris forceps (10 cm, curved, serrated)</td><td>FST</td><td>14007-14</td><td></td></tr><tr><td>iris scissors (11 cm, straight, tungsten carbide)</td><td>FST</td><td>14501-14</td><td></td></tr><tr><td>Isofluran OP Tisch, electrically heated, sm&lt;br/&gt; Outer dimensions: 257mm x110 mm x 18 mm.&lt;br/&gt; Heating area: 190 mm x 90 mm&lt;br/&gt; The removal of the isoflurane escaping&lt;br/&gt; the breathing mask is downwards in compliance with the&lt;br/&gt; regulations</td><td>Biomedical Instruments (Univentor)</td><td>-</td><td></td></tr><tr><td>isoflurane (Attane)</td><td>JD medical</td><td></td><td>inhalation anesthesia</td></tr><tr><td>LED RGB lights</td><td>Cameo</td><td>CLQS15RGBW</td><td>LEDs 2 x 15 W</td></tr><tr><td>Light microscope Basic DM E</td><td>Leica</td><td>-</td><td>4x (N.A. 0.1 &amp;infin;/-), 10x (N.A. 0.22 &amp;infin;/0.17), 40x (N.A. 0.65 &amp;infin;/0.17), 100x (N.A. 1.25 &amp;infin;/0.17) objectives</td></tr><tr><td>micropipette puller</td><td>Science-Products</td><td>P-97</td><td></td></tr><tr><td>Mosquito hemostatic forceps (12.5 cm, curved)</td><td>FST</td><td>13010-12</td><td></td></tr><tr><td>Nickel grids, 200 mesh</td><td>Ted Pella</td><td>1GC200</td><td></td></tr><tr><td>Osmium (VIII)-oxid</td><td>Degussa</td><td>73219</td><td></td></tr><tr><td>Propylene oxide</td><td>Fluka</td><td>82320</td><td></td></tr><tr><td>razor blades</td><td>Schick</td><td>87-10489</td><td></td></tr><tr><td>Sodium pentobarbital (Narcoren)</td><td>Merial GmbH</td><td>-</td><td></td></tr><tr><td>TC01mR 1-Channal temperature controller with feedback</td><td>Biomedical Instruments (Univentor)</td><td>-</td><td></td></tr><tr><td>Technovit 4004 two components glue</td><td>Kulzer</td><td></td><td></td></tr><tr><td>Telemacrodevice</td><td>Canon</td><td>-</td><td>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><td>Thermopuller P-97</td><td>Sutter Instruments</td><td>-</td><td></td></tr><tr><td>thin vibrating razor blade device</td><td>Krup</td><td>-</td><td>with Szabo thin blades</td></tr><tr><td>toluidine blue</td><td>Sigma-Aldrich</td><td>89640</td><td></td></tr><tr><td>Transmission electron microscope C20</td><td>Phillips</td><td>-</td><td>up to 200 kV</td></tr><tr><td>Tygon 6/4 Tubing material for connection of all parts&lt;br/&gt; Outer diameter: 6 mm&lt;br/&gt; Inner diameter: 4 mm&lt;br/&gt; Wa&lt;br/&gt; ll thickness: 1 mm</td><td>Biomedical Instruments (Univentor)</td><td>-</td><td></td></tr><tr><td>Ultracut E</td><td>Reichert-Jung</td><td>-</td><td>ultramicrotome</td></tr><tr><td>Univentor Scavenger</td><td>Biomedical Instruments (Univentor)</td><td>8338001</td><td></td></tr><tr><td>Vannas scissors (8 cm, straight)</td><td>FST</td><td>15009-08</td><td></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 or through a mask to the animal (Figure 1A). The scavenger collects and inactivates any surplus isoflurane gas that may be produced, thus providing a safe working environment. The gas is collected and passed through active coal in a cartridge (Figure 1A).
An optimized set of instruments (Figure 1B) allows scientists to perform surgery quickly on pregnant mice. A simple paraffin film with hydrophobic properties prevents the abdominal mucosa from drying after resection (Figure 1B). The uterine horns harboring the embryos are exposed onto paraffin film and the embryos are numbered in the way shown in Figure 1C. Once localized, the embryos are adjusted in the correct position for injection of the virus via a thin capillary (Figure 1D). The injection is visually controlled by observing the diffusion shape of the penetrating dye contained in the injected virus mixture (Figure 1D). After in utero injection, the uterine horns are placed back in the abdominal cavity to allow the development of the injected embryos until the stage (e.g., until birth and reaching postnatal day 24) that is needed for the experiment (see, for example, Lutz et al.5,6 and Kraus et al.7). For studies on synaptic plasticity, late developmental or adult stages are recommended.
After a transcardial perfusion of the animals at the desired stage, the nervous tissue (for example, the brain and spinal cord in Figure 2) is placed and oriented in a transparent plastic frame with coordinates and embedded in agarose (Figure 2A,B). After cutting, every section is imaged (Figure 2C,E,G) and then osmicated. After osmication and incubation in 70% or 80% ethanol, the sections are imaged again by using interference light telemacrography8,9,10. The tissue sections must be placed in a dull black surrounding to minimize scattered radiation during the required high-intensity illumination. Fiber tracts and different areas of the tissue reflect iridescent colors that contrast the dark tissue background, and a specifically colored pattern of the tissue surface emerges (Figure 2D,F,H). All iridescent images are superimposed with the nonosmicated images and, together with the projected coordinates of the embedding step, they generate navigation maps of the tissue topography. Next, the tissue is further dehydrated and embedded into resin. Based on the navigation maps, areas of interest are chosen and further processed for TEM-as examples, the hippocampus (stratum lucidum), cerebellum (granule cell layer), and spinal cord (dorsal funiculus) are shown in Figure 3A1-C4.
In the hippocampus and cerebellum, mossy fiber synapses are known to exhibit unique ultrastructural characteristics when compared to other synapses, including large presynaptic boutons (Figure 3A1-B5). They can be easily localized with the help of the iridescent navigation maps established during the previous processing steps. In their cross-section profiles, the boutons contain a vast number of vesicles and mitochondria and very often enclose several dendritic spines (Figure 3A4,A5,B4,B5). TEM images of these profiles allow a quantification of cross-sectioned synaptic bouton areas, numbers of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, and numbers of contact sites.
In the spinal cord, the dorsal funiculus contains many axonal fibers which are myelinated by oligodendroglia. On transversal sections, the dorsal funiculus can be found between both posterior horns of the spinal cord on transversal sections (Figure 3C1,C2). On TEM images, cross-sectioned myelinated axons appear round and the myelin sheaths surrounding the axons are organized in lamellae (Figure 3C3,C4). A quantification of cross-sectioned axonal areas, including the thickness of myelin sheaths and the number of myelin lamellae, as well as a quantification of cross-sectioned mitochondria profile areas can be carried out.
The prepared atlas and the micrographic TEM images of the ultrastructural parameters are not only helpful for a comparison of morphological neuroplasticity upon different conditions of treatment, but also when a comparison between parameters within the same area is required (e.g., a comparison between different type of synapses in the hippocampus18 and between equivalent structures of different species [Figure 4]).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59084/59084fig1.jpg" /> 
Figure 1: Preparation for in utero transduction. (A) Isoflurane anesthesia setup: 1) induction chamber for initial anesthesia; 2) pump; 3) anesthesia unit; 4) routing switch valve; 5) anesthesia supply tubing; 6) scavenger unit; 7) breathing mask; 8) heated surgical table; 9) binocular stereoscope with light source; 10) control unit. (B) Surgery equipment: 1) electric shaver; 2) eye lubricant; 3) fixation tapes; 4) sanitary pads; 5) iris forceps (10 cm, curved, serrated); 6 and 7) iris scissors (11 cm, straight, tungsten carbide); 8) Dumont tweezers (#3, 12 cm, straight, 0.2 mm x 0.12 mm); 9) Vanna&#39;s scissors (8 cm, straight); 10 and 11) micro-mosquito hemostatic forceps (12.5 cm, curved); 12) paraffin film; 13) micro spoon; 14) cotton swabs; 15 and 16) Halsted&#39;s mosquito hemostatic forceps (12.5 cm, straight); 17 and 18) sutures (size 6-0 and 3-0); 19) syringe; 20) aspirator tube assemblies for calibrated microcapillary pipettes. (C) Scheme of a systematic numbering of embryos within the uterine horns at embryonic day (E) 14.5: L = left; R = right. (D) Strategies of injection: into the first and second brain ventricles (Ive and IIve, respectively), fourth ventricle (IVve), and spinal cord. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59084/59084fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59084/59084fig2.jpg" /> 
Figure 2: Documentation of isolated tissue by telemacrography. (A and B) Embedding of a brain and a spinal cord in agarose, using cuvettes with a coordinate system (grids). After cutting, the sections are subjected to telemacrography and interference reflection light imaging. (C-F) Transversal and sagittal sections of the brain. Scale bars = 5 mm; co = cortex; st = striatum; di = diencephalon; me = mesencephalon; hi = hippocampus; ped = pedunculi cerebri; ce = cerebellum; mo = medulla oblongata. (G and H) Transversal section of a cervical segment of the spinal cord. Scale bar = 1 mm; ahR = right anterior horn;&#160;ahL = left anterior horn; df = dorsal funiculus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59084/59084fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59084/59084fig3.jpg" /> 
Figure 3: Topographical and ultrastructural identification of plasticity parameters. (A1) Inverted interference light macrograph of the hippocampus. The region of the mossy fiber boutons within the stratum lucidum (sl) is highlighted. pcl = pyramidal cell layer; gcl = granule cell layer. Scale bar = 500 &#181;m. (A2) Light microphotograph (100x objective) of a semithin section (0.75 &#181;m) showing stratum lucidum. The asterisks indicate the regions of mossy fiber boutons that surround many dendritic islands (black arrowheads). Toluidine blue/OsO4 staining. Scale bar = 50 &#181;m. (A3) Transmission electron micrograph at a low magnification, showing mossy fiber boutons (asterisks) surrounding a dendrite (D). The rectangle includes a single mossy fiber bouton. Scale bar = 2 &#181;m. (A4) Transmission electron micrograph of the cross-section profile of a single mossy fiber bouton (MFB). Spines (blue) and the cross-section bouton area (violet) are highlighted. m = mitochondrion; s = spines. Scale bar = 200 nm. (A5) Synaptic vesicles inside an MFB. Scale bar = 100 nm. (B1) Inverted interference light macrograph of the cerebellum. The region of the granule cell layer (gcl) that contains the mossy fiber boutons is indicated. mcl = molecular layer; h = hilus. Scale bar = 500 &#181;m. (B2) Light microphotograph (100x objective) of a semithin section (0.75 &#181;m) showing a region of the granule cell layer that borders on Purkinje cells (PC). The asterisks indicate the regions of mossy fiber boutons that are surrounded by many cerebellar granule neurons (CGN). Toluidine blue/OsO4 staining. Scale bar = 50 &#181;m. (B3) Transmission electron micrograph at a low magnification, showing the area of MFBs. The rectangle includes a single MFB. Scale bar = 2 &#181;m. (B4) Transmission electron micrograph of the cross-section profile of a mushroom-like MFB within the granule cell layer of the cerebellum. The cross-section bouton area (violet) and vesicle-free spines (blue) are highlighted. Scale bar = 1 &#181;m. (B5) Mitochondria (violet) and synaptic vesicles inside an MFB. Scale bar = 150 nm. (C1) Inverted interference light macrograph of the spinal cord. The region of the dorsal funiculus (df) that contains heavily myelinated axons is highlighted. ahR and ahL = right and left anterior horn, respectively; phR and phL = right and left posterior horn, respectively. Scale bar = 500 &#181;m. (C2) Light microphotograph (100x objective) of a semithin section (0.75 &#181;m) showing the dorsal funiculus that is enriched with heavily myelinated axons (ax) that are clearly distinguishable from nonmyelinated fibers (asterisks). ct = connective tissue. Toluidine blue/OsO4 staining. Scale bar = 50 &#181;m. (C3) Transmission electron micrograph of cross-section profiles of myelinated and nonmyelinated axons (ax, violet). my = myelin sheaths. Scale bar = 200 nm. (C4) The rectangle shows high-magnified, heavily myelinated axons with myelin lamellae and nonmyelinated fibers. The microtubules (mt) and mitochondria (m) within the axonal fibers are visible. Scale bar = 50 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59084/59084fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59084/59084fig4.jpg" /> 
Figure 4: Examples of ultrastructural plasticity parameters in different animal models. (A) Mossy fiber boutons in the cerebellum&#160;of a shark. Scale bar = 400 nm. (B) Mossy fiber bouton in the hippocampus of a rhesus macaque. Scale bar = 200 nm. (C) Neuromuscular junction of a cat. Scale bar = 300 nm. (D) Synaptic boutons in the pars reticularis substantia nigra of a phalanger. The asterisks indicate postsynaptic density (PSD; in inset: black arrowheads). The rectangle shows a magnified PSD. Scale bar = 400 nm. (E) A nonmyelinating Schwann cell (SC) surrounding many axonal fibers in the spinal ganglion of a mouse. In the vicinity, heavily myelinated fibers are visible. Scale bar = 300 nm. For all panels: ax = axon; D = dendrite; m = mitochondrion; MFB = mossy fiber bouton; MF = mossy fiber; my = myelin sheaths; s = spine; sa = spine apparatus; sv = synaptic vesicles. Contact sites (postsynaptic density) are indicated with asterisks. Cross-section areas are highlighted in violet and blue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59084/59084fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.8K Views.  Ruhr University Bochum. Onze gecombineerde aanpak helpt te beantwoorden hoe ultrastructurele neuroplasticiteit parameters veranderen na vroege ontwikkelingsinterventies aan het ontluikende zenuwstelsel in de baarmoeder. Het belangrijkste voordeel van onze methode is het verwerven van beelden met hoge resolutie van macro-meso-micro-en nanostructuren in het vooraf gedefinieerde coördinatensysteem binnen een weefselatlas. De implicatie van onze methode is het translationele therapeutische potentieel voor aangeboren ne...

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Neuroscience

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Medicine

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

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

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