The work was supported by NIH grants R01NS098804 (A.E.W.), F31NS113394 (U.C.), and Duke University&#39;s Summer Neuroscience Program (D.G.).

NOTE: All procedures were performed under protocols approved by Duke University Institutional Animal Care and Use Committee (IACUC).
1. DNA preparation for in vivo electroporation or IVE (1 day before surgery)
<ol>
	<li>Gather the following materials: purified DNA (0.5-25 &#181;g per animal), 3 M sodium acetate, ethanol, Fast Green dye, ultrapure distilled water, phosphate buffer solution (PBS) (see the&#160;Table of Materials). 
	&#8203;NOTE: For DNA, a construct expressing green fluorescent protein (GFP) under a human ubiquitin promoter was obtained from Addgene (FUGW, https://www.addgene.org/14883/). Any construct expressing GFP or another fluorescent protein under the control of a ubiquitous promoter should work. CGN-specific labeling with this technique is not dependent on the construct, but rather on the electroporation.</li>
	<li>Prepare DNA for electroporation by mixing the desired amount of DNA, 10% by volume of 3 M sodium acetate and 250% by volume of 100% ice-cold ethanol. Note that the DNA will precipitate out of solution immediately.</li>
	<li>Continue to precipitate the DNA mixture overnight at -20 &#176;C or for an hour at -80 &#176;C.</li>
	<li>Pellet precipitated DNA in a tabletop centrifuge at &#62;16,000 &#215; g and wash twice with 70% ethanol.</li>
	<li>Allow the DNA pellet to dry completely, and reconstitute in a 1x PBS + 0.02% Fast Green solution.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62568/62568fig01.jpg" /> 
Figure 1: Limiting injection depth to 1.5 mm using a spacer. (A) A 11.2 mm segment is cut off a loading pipette using a razor blade. (B) The spacer is fitted onto the tip of the Hamilton syringe (total length is 1.27 cm or 0.5 in) and secured with either adhesive or parafilm. The exposed tip should be 1.5 mm in length. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. In vivo electroporation of granule neuron progenitors in seven-day old postnatal mice
NOTE: All electroporation surgeries were performed in a sterile and highly ventilated surgical suite, and all personnel wore complete personal protective equipment including gloves, face mask, hair bonnet, gown, and shoe covers. Alternatively, the surgeries can be performed in a ventilated and sterile hood.
<ol>
	<li>Gather the following materials: DNA for electroporation, small surgical scissors, small surgical tweezers, customized Hamilton syringe, cotton-tip applicator, heating pad, betadine, 70% ethanol, 1x PBS, parafilm, tissue adhesive (n-butyl-ester cyanoacrylate), isoflurane, electroporator, and tweezer-type electrodes (see the Table of Materials).</li>
	<li>Cut a spacer from a sterilized loading tip to fit over the Hamilton syringe to limit the injection depth to 1.5 mm (Figure 1A,B). Secure the spacer with adhesive or parafilm.</li>
	<li>Anesthetize the P7 pup in an isoflurane chamber at a delivery rate of 0.8 L/min. Confirm full anesthesia by monitoring the animal for decreased respiration and a lack of a toe or tail pinch response (Figure 2A).</li>
	<li>Once the animal is fully anaesthetized, place the pups on a pedestal fitted with a nose cone, delivering constant 4% isoflurane at a delivery rate of 0.8 L/min. Clean the top of the pup&#39;s head 3 times with a sterile swab of betadine then 70% ethanol, alternating between the two, to prepare the site.&#160;Allow the solution to dry before proceeding.</li>
	<li>Using a pair of sterilized scissors, make a small incision with one cut that spans the distance from the top to the base of the ears to reveal the hindbrain (Figure 2B).</li>
	<li>Locate the cerebellum (Figure 2C), insert the exposed tip of the Hamilton syringe through the skull, perpendicular to the brain, and inject 1.5 &#181;L of DNA mixture into the cerebellar parenchyma by slowly pushing the back plunger of the syringe. After delivery of the DNA mixture, slowly pull the needle back to prevent back spills, and allow the DNA solution to diffuse for 30 s.</li>
	<li>Turn off the isoflurane, and place the pup on a 37 &#176;C heating pad. Prepare the tweezer-type electrode for electroporation by dipping both ends into sterile 1x PBS. 
	NOTE: Wetting the tweezer-type electrode will prevent contact burns on the skin of the pup during administration of the electrical pulses.</li>
	<li>Orient the tweezer-electrode above the site of injection with the plus end facing downwards and the negative end above the animal&#39;s head (Figure 2D). Administer five electrical pulses from the electroporator with the following settings: 50 ms, 130 V, and 950 ms inter-pulse interval. 
	NOTE: If needed, perform a test injection to ensure that the injection site is located on the cerebellar vermis (Figure 2E).</li>
	<li>Pinch the incision closed, and seal the wound with a non-toxic n-butyl-ester cyanoacrylate tissue adhesive. Clean the wound with 70% ethanol as any trace amount of blood increases the likelihood of parental infanticide and cannibalism.</li>
	<li>Allow the animal to recover on a 37 &#176;C heating pad before returning the pup to the dam. Monitor the pup(s) every 30 min for at least 2 h after the surgery to ensure full recovery. 
	&#8203;NOTE: Infanticide by either parent is quite common. To prevent cannibalism, house the sire in a different cage before starting the electroporation, and always return cleaned and recovered pups (i.e., no bloodstain, fully mobile) to the original cage on the original bedding. Pups can also be wiped with droppings from the original cage to minimize the smell of blood. The use of a surrogate dam may be necessary if the original dam continues to cannibalize her pups.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62568/62568fig2v2.jpg" /> 
Figure 2: In vivo cerebellar electroporation of granule neuron progenitors in P7 wildtype mouse pups. (A) Pups are anesthetized with 4% isoflurane delivered at a rate of 0.8L/min&#160;to ensure anesthesia throughout the injection of the DNA solution. Isoflurane is delivered at a rate of 0.8 L/min. (B) After sterilizing the mouse 3 times with betadine and 70% ethanol, an incision is made that spans the distance of the ears, revealing the hindbrain. (C) A magnified image of a white demarcation on the cranium, a landmark for the injection site. DNA construct should be injected within 1 mm above the mark; dotted lines outline the demarcation, and black arrow denotes the injection site. The ridges of the cerebellar vermis may be visible and can be useful for finding the injection site. (D) Tweezer-type electrode orientation for efficient electroporation. Plus (+) end must be oriented downwards to pull negatively charged DNA into the cerebellar parenchyma prior to administration of electrical pulses. (E) Test injection of 1 &#181;L of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Immunohistochemistry of electroporated CGNs
<ol>
	<li>Gather the following materials: isoflurane, 1x PBS, 4% paraformaldehyde (PFA), 30% sucrose, normal goat serum, non-ionic detergent, glass slides, glass coverslips, nail polish, mounting media, Hoechst nuclear dye, and appropriate primary and secondary antibodies (see the&#160;Table of Materials).</li>
	<li>Anesthetize the experimental animal with isoflurane, and confirm full anesthesia with a toe and tail pinch.</li>
	<li>Perform a trans-cardial perfusion by slowly injecting 1x PBS and 4% PFA into the left ventricle of the animal&#39;s heart. Allow the blood to drain from the animal by cutting the vena cava.</li>
	<li>Fix the brain overnight by submerging it in 4% PFA at 4 &#176;C. On the following day, quickly rinse the brain with 1x PBS, and transfer the brain into 30% sucrose in 1x PBS for cryoprotection for at least 24 h.</li>
	<li>If necessary, slice the brain in half along the rostral-caudal axis, and confirm expression of the transfected reporter construct using an upright fluorescent dissecting microscope. 
	NOTE: Keep the brain submerged in 1x PBS in a small dish to prevent it from drying out.</li>
	<li>Mount the brain on a freezing microtome, slice 25 &#181;m sagittal sections, and allow sections to unfold in a 1:1 mixture of 1x PBS and glycerol. 
	NOTE: Sections can be stored in this cryoprotectant solution at -20 &#176;C for long-term storage.</li>
	<li>Wash sections three times in 1x PBS for 10 min each to remove cryoprotectant, and block the tissue in 1x PBS + 10% normal goat serum + 0.2% non-ionic detergent on an orbital shaker at room temperature for 1 h.</li>
	<li>Prepare primary antibody solution: 1x PBS, 10% normal goat serum, 0.2% non-ionic detergent, and anti-GFP antibody, and centrifuge the solution for 5 min at &#62;16,000 &#215; g. Incubate sections in the antibody solution at 4 &#176;C on an orbital shaker for 48 h.</li>
	<li>Wash off primary antibody solution for 15 min five times with 1x PBS + 0.2% non-ionic detergent.</li>
	<li>Prepare secondary antibody solution: 1x PBS, 10% normal goat serum, 0.2% non-ionic detergent, and an appropriate secondary antibody to detect GFP; centrifuge the solution at &#62;16,000 &#215; g. Incubate sections in the antibody solution on an orbital shaker at room temperature for 2-3 h. Protect the sections from light exposure to prevent bleaching.</li>
	<li>Wash off secondary antibody solution three times with 1x PBS + 0.2% non-ionic detergent for 15 min each time. Incubate sections in 1x PBS + Hoechst for 5 min to stain nuclei.</li>
	<li>Wash off Hoechst solution with 1x PBS + 0.2% non-ionic detergent and mount onto glass slides. Cover the sections with mounting media, coverslip the slides, and seal the slide with nail polish to prevent evaporation.</li>
</ol>
4. Morphological analyses of CGNs - three-dimensional (3D) reconstruction and surface area and cellular volume
<ol>
	<li>Image single electroporated CGNs on a confocal microscope at 63x objective with a 2x zoom, taking z-stack images at 0.5 &#181;m per stack. Image one cell per image window to allow for easy image analysis and reconstruction.</li>
	<li>Install the Simple Neurite Tracer plug-in for FIJI using the following link (https://imagej.net/Simple_Neurite_Tracer:_Basic_Instructions) to easily and efficiently trace the structure of electroporated CGNs in three-dimensional (3D) space. 
	NOTE: There is an updated version of the plug-in (https://imagej.net/SNT).</li>
	<li>Analyze neurite length and dendritic claw formation in a blinded manner using Simple Neurite Tracer. Upload single-channel z-stack images of electroporated CGNs onto FIJI, and click on Plugins | Segmentation | Simple Neurite Tracer (Figure 3D).</li>
	<li>Access the drop-down menu, and select Create New 3D Viewer (Figure 3D).</li>
	<li>Scroll to the base of a dendrite, where it connects to the cell soma and start a path by clicking on the junction. Manually trace the path by clicking through the sections where the cell-fill signal is brightest, pressing [y] to keep the trace. Trace until the end of the dendrite if it does not contain a claw or until the base of the claw and confirm the path by pressing [f] (Figure 4D).</li>
	<li>Next, trace the claw by starting a path at the base of the structure and tracing until the end of the longest neurite. Trace secondary and tertiary branches by holding down [ctrl] on Windows or [alt] on a Mac OS and clicking the path. Confirm the path by pressing [f].</li>
	<li>Observe that measurements for the traces are visible on a separate window; add up all of the measurements of the claw branches (primary, secondary, tertiary) to obtain the total length for each claw.</li>
	<li>For analyzing surface area and cellular volume of electroporated CGNs, download Imaris cell analysis software (https://imaris.oxinst.com/). 
	NOTE: FIJI can also be used to reconstruct cells in 3D from z-stack images using readily available and free plug-ins. Additionally, there is a volumetric rendering feature in Simple Neurite Tracer but Imaris was used for the reasons outlined below.</li>
	<li>Upload z-stack image of an electroporated CGN onto Imaris. Access the 3D-reconstruction toolkit by pressing Surpass.</li>
	<li>To reconstruct the CGN, press Surfaces, and select a region of interest that encompasses the entirety of the cell within the image window. Once finished, press the blue forward arrow at the bottom right corner under Create.</li>
	<li>If the image contains multiple channels for different signals, select the channel containing the electroporated CGN, and press the blue forward arrow.</li>
	<li>Using the slidebar, set a desired threshold that most accurately fits the signal of the electroporated cell. Zoom in closer to the surface of the cell to accurately determine the threshold. Once finished, press the double green arrow to reconstruct the cell and obtain the surface area and volume size from the metadata.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62568/62568fig3v2.jpg" /> 
Figure 3: Immunohistochemical analysis and three-dimensional reconstruction of electroporated granule neurons. P7 CD-1 mice were electroporated with a construct expressing GFP. Brains were collected and subjected to immunohistochemistry, confocal microscopy, and 3D-reconstruction for morphological analysis. (A) Timeline from electroporation to image processing of a 10-DPI mouse. (B) Maximum projection image of a sagittal cross-section of electroporated cerebellum 10-DPI; white lines demarcate cerebellar layers, and scale bar is 25 &#181;m. (C) Maximum projection image of a single electroporated granule neuron 10-DPI and the corresponding 3D trace, scale bar is 10 &#181;m. (D) 3D-reconstructions were generated using the FIJI plugin Simple Neurite Tracer. All measurements were traced through the z-stack, following the cell-fill signal. Shaft and claw measurements were traced separately for every dendrite; dotted line denotes portion of dendrite within the current plane. Abbreviations: 3D = three-dimensional; GFP = green fluorescent protein; DPI = days post-injection; PSD-95 = postsynaptic density protein 95; GNPs = granule neuron progenitors; PFA = paraformaldehyde. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest.

Cerebellar granule neurons are the most abundant neurons in the mammalian brain, making up almost 60-70% of the total neuron population in the rodent brain1,14. The cerebellum has been extensively utilized to elucidate mechanisms of cellular proliferation, migration, dendrite formation, and synapse development6,9,10,11,<sup c...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62568">Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development</a>. A figure was updated.
Figure 2 was updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62568/62568fig02.jpg" /> 
Figure 2: In vivo cerebellar electroporation of granule neuron progenitors in P7 wildtype mouse pups. (A) Pups are anesthetized with 4% isoflurane delivered at a rate of 0.8L/min&#160;to ensure anesthesia throughout the injection of the DNA solution. Isoflurane is delivered at a rate of 0.8 L/min. (B) After sterilizing the mouse 3 times with betadine and 70% ethanol, an incision is made that spans the distance of the ears, revealing the hindbrain. (C) A magnified image of a white demarcation on the cranium, a landmark for the injection site. DNA construct should be injected within 1 mm above the mark; dotted lines outline the demarcation, and black arrow denotes the injection site. The ridges of the cerebellar vermis may be visible and can be useful for finding the injection site. (D) Tweezer-type electrode orientation for efficient electroporation. Plus (+) end must be oriented downwards to pull negatively charged DNA into the cerebellar parenchyma prior to administration of electrical pulses. (E) Test injection of 1 &#181;L of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62568/62568fig2v2.jpg" /> 
Figure 2: In vivo cerebellar electroporation of granule neuron progenitors in P7 wildtype mouse pups. (A) Pups are anesthetized with 4% isoflurane delivered at a rate of 0.8L/min&#160;to ensure anesthesia throughout the injection of the DNA solution. Isoflurane is delivered at a rate of 0.8 L/min. (B) After sterilizing the mouse 3 times with betadine and 70% ethanol, an incision is made that spans the distance of the ears, revealing the hindbrain. (C) A magnified image of a white demarcation on the cranium, a landmark for the injection site. DNA construct should be injected within 1 mm above the mark; dotted lines outline the demarcation, and black arrow denotes the injection site. The ridges of the cerebellar vermis may be visible and can be useful for finding the injection site. (D) Tweezer-type electrode orientation for efficient electroporation. Plus (+) end must be oriented downwards to pull negatively charged DNA into the cerebellar parenchyma prior to administration of electrical pulses. (E) Test injection of 1 &#181;L of a 0.02% Fast Green dye shows injection is localized to the middle of the cerebellar vermis between lobules 5-7. <a href="https://www.jove.com/files/ftp_upload/62568/62568fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62568">Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development</a>. A figure was updated.

Erratum: Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Brain development is a prolonged process that extends from embryogenesis into postnatal life. During this time, the brain integrates a combination of intrinsic and extrinsic stimuli that sculpt the wiring of synapses between dendrites and axons to ultimately guide behavior. The rodent cerebellum is an ideal model system to study how synapses develop because the development of a single neuron type, the cerebellar granule neuron (CGN), can be tracked as it transitions from a progenitor cell to a mature neuron. This is due, in part, to the fact that a majority of the cerebellar cortex develops postnatally, which allows for easy genetic manipulation and cell labeling after birth1.
In mammals, CGN differentiation begins at the end of embryonic development when a subset of proliferative cells in the hindbrain migrates over the rhombic lip to form a secondary germinal zone on the surface of the cerebellum2,3,4. Although they are fully committed to a granule neuron progenitor (GNP) identity, these cells continue to proliferate within the outer portion of the external granule layer (EGL) until postnatal day 14 (P14). Proliferation of this layer results in a massive expansion of the cerebellum as these cells give rise exclusively to CGNs5. Once newborn CGNs exit the cell cycle in the EGL, they migrate inwards towards the internal granule layer (IGL), leaving behind an axon that will bifurcate and travel in the molecular layer of the cerebellum, forming parallel fibers that synapse onto Purkinje cells6. The position of these fibers within the molecular layer is dependent on the timing of cell-cycle exit.
CGNs that differentiate first leave their parallel fibers towards the bottom of the molecular layer, whereas the axons of CGNs that differentiate later are clustered at the top7,8. Once the CGN cell bodies reach the IGL, they begin to elaborate dendrites and form synapses with nearby inhibitory and excitatory neurons. The mature dendritic tree of a CGN exhibits a stereotyped architecture with four main processes. Over the course of CGN maturation, the structures at the end of these dendrites form a claw that becomes enriched with postsynaptic proteins9,10. These specialized structures, called dendritic claws, contain the majority of the synapses onto granule neurons and are important for receiving both excitatory inputs from mossy fiber innervations originating from the pons, as well as inhibitory inputs from local Golgi cells. Once fully configured, the synaptic connections of CGNs allow these cells to relay inputs from pre-cerebellar nuclei to Purkinje cells, which project out of the cerebellar cortex to the deep cerebellar nuclei.
In vivo postnatal electroporation of GNPs is advantageous over other labeling-based methods, such as viral infection and generation of transgenic mouse lines, because the expression of desired constructs can be achieved on a fast timeline, and the method targets a small population of cells, useful in studying cell-autonomous effects. This method has been used in prior studies to study morphological development of CGNs; however, these studies have focused on either a single time point or a short window of time9,10,11,12,13. This labeling method was paired with image analysis to document the changes in CGN morphology that occur across the entire time course of CGN differentiation over the first three weeks of postnatal life. These data reveal the dynamics of CGN dendrite development that underlie construction of cerebellar circuits.

<table><tbody><tr><td>Betadine</td><td>Purdue Production</td><td>67618-150-17</td><td></td></tr><tr><td>Cemented 10 &amp;micro;L needle</td><td>Hamilton</td><td>1701SN (80008)</td><td>33 gauge, 1.27 cm (0.5 in), 4 point style</td></tr><tr><td>Chicken anti-GFP</td><td>Millipore Sigma</td><td>AB16901</td><td>Our lab uses this antibody at a 1:1000 concentration</td></tr><tr><td>Cotton-tip applicator</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-chicken Cy2</td><td>Jackson ImmunoResearch</td><td>703-225-155</td><td>Our lab uses this antibody at a 1:500 concentration</td></tr><tr><td>Ethanol (200 proof)</td><td>Koptec</td><td>V1016</td><td></td></tr><tr><td>Electroporator ECM 830</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td></td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>FUGW plasmid</td><td>Addgene</td><td>14883</td><td></td></tr><tr><td>Glass slides</td><td>VWR</td><td>48311-703</td><td>Superfrost plus</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Heating pad</td><td>Softheat</td><td></td><td></td></tr><tr><td>Hoescht 33342 fluorescent dye</td><td>Invitrogen</td><td>62249</td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>07-893-1389</td><td></td></tr><tr><td>Micro cover glass</td><td>VWR</td><td>48382-138</td><td></td></tr><tr><td>Nail polish</td><td>Sally Hansen</td><td>Color 109</td><td></td></tr><tr><td>Normal goat serum</td><td>Gibco</td><td>16210064</td><td></td></tr><tr><td>O.C.T. embedding compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Olympus MVX10 Dissecting Scope</td><td>Olympus</td><td>MVX10</td><td></td></tr><tr><td>P200 pipette reach tip</td><td>Fisherbrand</td><td>02-707-138</td><td>Used for needle spacer</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>PBS pH 7.4 (10x)</td><td>Gibco</td><td>70011-044</td><td></td></tr><tr><td>Simple Neurite Tracer</td><td>FIJI</td><td></td><td>https://imagej.net/Simple_Neurite_Tracer:_Basic_&lt;br/&gt; Instructions</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Surgical tools</td><td>RWD Life Science</td><td></td><td>Small scissors and tweezers</td></tr><tr><td>Triton X-100</td><td>Roche</td><td>11332481001</td><td>non-ionic detergent</td></tr><tr><td>Tweezertrodes</td><td>BTX Harvard Apparatus</td><td>45-0489</td><td>5 mm, platinum plated tweezer-type electrodes</td></tr><tr><td>Ultrapure distilled water</td><td>Invitrogen</td><td>10977-015</td><td></td></tr><tr><td>Vectashield mounting media</td><td>Vectashield</td><td>H1000</td><td></td></tr><tr><td>Vetbond tissue adhesive</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Zeiss 780 Upright Confocal</td><td>Zeiss</td><td>780</td><td></td></tr></tbody></table>

utilizing in vivo postnatal electroporation to study cerebellar granule neuron morphology and synapse development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62568/62568fig04.jpg" /> 
Figure 4: Analysis of granule neuron morphology during cerebellar development. (A) Maximum projection images of electroporated CGNs from 3-DPI to 14-DPI (postnatal age P10 to P21), nuclei (blue) and GFP (green); arrowheads indicate individual dendrite, and scale bar is 10 &#181;m. (B) Average number of dendrites. (C) Average dendrite le...

Watch this Scientific Journal Video about Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development at JoVE.com

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

utilizing-vivo-postnatal-electroporation-to-study-cerebellar-granule

Research

JoVE Journal

Neuroscience

2.5K Views.  Duke University Medical Center. Het protocol bestudeert cerebellaire korrelneuronen in verschillende stadia van ontwikkeling om de belangrijkste morfologische groei te visualiseren. De voordelen van de techniek zijn celspecifieke targeting, snelle expressie van getransfecteerde constructen en schaarse labeling van cellen die de studie van celautonome effecten mogelijk maakt. Begin met het snijden van een segment van 11,2 millimeter uit een punt van een laadpipet en plaats het gesneden deel over de punt van de Hamilton-spuit...

Video: Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Developmental Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Biology

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 ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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).

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 ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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, ...

Visualizing the Synaptogenesis of Cerebellar Granule Neurons at Different Developmental Stages

Source: Chan, U.,&#160;et al., Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development.&#160;J. ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

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