Name: Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons (Video) | JoVE Journal
Uploaded: 2018-04-20T15:00:00.000+00:00
Duration: 9 min 50 s
Description: Learn ex utero electroporation and organotypic slice cultures to visualize migrating GABAergic interneurons in embryonic mouse brains. This low-cost method enables live-imaging of neuronal development for neurodevelopmental research.

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5 minutes

This work was supported by operating grants from the Savoy Foundation and the CURE Epilepsy Foundation and by equipment grants from the Canadian Foundation for Innovation to E.R (confocal microscope) and G.H (spinning disk confocal microscope). E.R. receives a career award from the Fonds de recherche du Qu&#233;bec-Sant&#233; (FRQ-S; Clinician-scientist Award) as well as from the Canadian Institutes for Health Research (CIHR; Young Investigator Award). G.H. is a senior scholar of the FRQ-S. L.E is the recipient of the Steriade-Savoy postdoctoral training award from the Savoy Foundation, the CHU Sainte-Justine Foundation postdoctoral training award and the FRQ-S postdoctoral training award, in partnership with the Foundation of Stars. This project has been made possible by Brain Canada through the Canada Brain Research Fund, with the financial support of Health Canada, awarded to L.E.

All experiments involving animals were approved by the Comit&#233; Institutionnel des Bonnes Pratiques avec les Animaux de Recherche (CIBPAR) at the CHU Sainte-Justine Research Center and were conducted in accordance with the Canadian Council on Animal Care&#39;s guide to the Care and Use of Experimental Animals (Ed. 2).
The protocol described here was optimized for electroporation of embryos at embryonic day (e) 13.5, at a time when MGE-derived INs are actively generated, before the peak of CGE-derived INs production45,46. Furthermore, to bias the electroporation towards GABAergic INs, we use a promoter selectively expressed in INs (for instance the Dlx5/6 promoter with its minimal enhancer)47.
1. Preparation of Solutions for Electroporation and Organotypic Slice Cultures
<ol>
	<li>Prepare 125 mL of sterile culture medium.
	<ol>
		<li>Measure 125 mL of regular neuron-specific culture medium (see Table of Materials for formulation) in a sterilized bottle in a previously UV-sterilized biosafety cabinet sprayed with 70% ethanol. Add 2.25 mL of 50x serum-free neuron-specific supplement, 1.75 mL of 200mM glutamine (final concentration of 0.5 mM) and 6.25 mL of heat-inactivated horse serum previously aliquoted under sterile conditions. Mix thoroughly, aliquot in 15 mL sterile conical tubes, and store at 4 &#176;C. 
		NOTE: Prepared culture medium can be stored for up to 3 weeks at 4 &#176;C.</li>
		<li>Divide a 100X stock solution of Botteinstein&#39;s N-2 formulation48 into 150 &#181;L aliquots under sterile conditions and freeze at -20&#176;C until use.</li>
	</ol>
	</li>
	<li>Prepare 1 L of sterile artificial cerebrospinal fluid (ACSF).
	<ol>
		<li>Measure 800 mL of distilled water in a 1 L beaker. Add 25.67 g of sucrose, 5.08 g of sodium chloride (NaCl), 2.18 g of sodium bicarbonate (NaHCO3), 1.80 g of glucose, 0.19 g of potassium chloride (KCl), 0.15 g of monobasic anhydrous sodium phosphate (NaH2PO4), 1 mL of 1M stock CaCl2.2H2O and 2 mL of 1M stock MgSO4.7H2O. Stir to dissolve at room temperature. Add distilled water to reach a total volume of 1 L.</li>
		<li>Using a 0.22 &#181;m filter, filter the solution into a sterile bottle in a sterilized biosafety cabinet and store at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
	<li>Prepare a fresh solution of 4% agarose in ACSF before each experiment.
	<ol>
		<li>Measure 25 mL of previously prepared ACSF in a 50-mL sterile conical tube and add 1 g of low-melting point agarose.</li>
		<li>Heat for 45 s in a microwave oven. To avoid spilling, interrupt heating every 3 - 4 s when boiling starts, open the tube to let the pressure out, close it again and agitate manually to mix the agarose. Repeat until the agarose solution is homogeneous. Keep the agarose solution at 42 &#176;C during the remainder of the experiment to avoid solidification. 
		NOTE: Higher temperatures will damage brain tissues.</li>
	</ol>
	</li>
</ol>
2. Preparation of Plasmids for Injection
<ol>
	<li>Pull a glass microinjection pipette
	<ol>
		<li>Set the micropipette puller with the proper parameters, secure the glass capillary in the provided space and make sure it is centered with the filament.</li>
		<li>Press the pull button.</li>
		<li>Carefully remove the newly made microinjection pipettes from the heat puller and place in a box or clean petri dish until further use to avoid damaging the tip.</li>
	</ol>
	</li>
	<li>Set-up the biosafety cabinet with all the instruments needed for this experiment (see Figure 1A), generously spray all instruments in the biosafety cabinet with 70% ethanol and sterilize the instruments and environment with UV light for 15 - 20 min.</li>
	<li>During the sterilization step, thaw plasmids on ice (4 &#176;C).</li>
	<li>Measure 10 &#181;L of the plasmid from a stock solution (4 &#181;g/&#181;L) into a clean 1.5 mL centrifuge tube. Add 0.01% of Fast Green FCF. Mix gently, spin briefly and keep on ice until use. 
	NOTE: Maxi-prep DNA should be prepared according to the manufacturer&#39;s protocol using an endotoxin-free maxi-prep kit. DNA can be solubilized in TE buffer or nuclease-free H2O and the preparation of the plasmid with dye solution can, but does not need to be performed under sterile conditions. Plasmids from Maxi-prep should be aliquoted to avoid multiple freeze-thaw cycles. Aliquoted plasmids should be mixed with the dye no more that 2 h before use and should not be refrozen for further use.</li>
	<li>After sterilization, prepare the nano-injector as follows.
	<ol>
		<li>Choose one of the previously prepared glass microinjection pipette stored in a clean box or petri dish and use small tweezers to cut the tip of the pipette in a beveled way to achieve an outer diameter of roughly 15 &#181;m. 
		NOTE: The outer diameter given here is an approximate measure to give the user an idea of what we use in our experiments and has been optimized in order to facilitate the piercing of the skull for plasmid injection without damaging the brain, while allowing for the fluid loading and release of the DNA. The outer diameter can be measured by looking at the cut tip of the glass microinjection pipette apposed to a micrometric scale bar under a bright field microscope.</li>
		<li>Use a syringe to fill the micropipette with mineral oil from its unpulled end (to expel all the air).</li>
		<li>Insert the filled glass micropipette in the nano-injector by following the manufacturer&#39;s instructions.</li>
		<li>Empty 2/3rds of the glass micropipette (keeping enough oil to prevent air entry).</li>
	</ol>
	</li>
	<li>Carefully insert the prepared micropipette in the tube containing the plasmid/dye solution and fill the glass micropipette with the plasmid/dye solution.</li>
</ol>
3. Collection of Mouse Embryos from Pregnant Females
<ol>
	<li>Monitor breeding females daily to assess for vaginal plugging, preferably at the same time daily (early afternoon). Day e0.5 corresponds to the first day when a vaginal plug is observed. 
	NOTE: The experiments described here can be conducted in wild-type mice. However, to facilitate the identification of the MGE and to label all GABAergic INs (or specific sub-sets such as MGE-derived INs), transgenic animals can be used (e.g.: GAD67EGFP; Dlx5/6Cre with a Cre-reporter allele, etc.47,49). In this situation, the experimental plasmid injected should express another fluorophore (for instance mCherry or TdTomato) to allow visualization of the transfected INs (yellow) that can be compared with non-transfected INs (green).</li>
	<li>Sacrifice the female at embryonic day e13.5, by neck dislocation. 
	NOTE: Anesthetic agents given at the time of sacrifice may impact IN migration and survival50,51 and should be avoided.</li>
	<li>Collect embryos by C-section as follows.
	<ol>
		<li>Generously spray the female abdomen with 70% ethanol. Pull the abdominal skin up with a pair of sterilized forceps and, with the other hand, use sterilized surgical scissors to cut the skin from the abdomen.</li>
		<li>With a second pair of sterilized forceps and scissors, pull the abdominal fascia up and cut it while carefully avoiding the uterus.</li>
		<li>Using a third pair of sterilized forceps and scissors, pull the uterine horns and cut them out of the pelvic cavity. Place the dissected uterine horns in a sterile 60-mm petri dish filled with neural-based culture medium supplemented with amino acids, vitamins and inorganic salts (see Table of Materials for commercially available product).</li>
	</ol>
	</li>
	<li>In a sterile biosafety cabinet, use two pairs of fine tweezers (one in each hand) to dissect the embryos out of the placenta and isolate the heads by decapitation.</li>
	<li>Bevel-cut the tip of a sterile 3 mL plastic transfer pipette, aspirate the heads and transfer them in a new sterile 60-mm petri dish layered with solidified black wax and filled with the same neural-based supplemented culture medium as above. 
	NOTE: This step minimizes the transfer of contaminants (mouse hair, blood). The black wax is used to stabilize the head during dissection. Culture media do not need to be oxygenated during these procedures.</li>
</ol>
4. Intraventricular Plasmid Injections and Ex Vivo Electroporation of the MGE
NOTE: The following steps must be performed under sterile conditions in the previously prepared biosafety cabinet.
<ol>
	<li>Place the 60-mm petri dish layered with black wax and containing the decapitated heads in neural-based supplemented culture medium under the binoculars in the biosafety cabinet.</li>
	<li>Stabilize the head, the rostral part facing right, with fine tweezers with the left hand and use the nano-injector in the right hand to inject 1 - 2 &#181;L of the plasmid/dye solution into the right lateral ventricle. 
	NOTE: Co-expression experiments can be conducted by co-electroporating a rescue plasmid and a shRNA-expressing plasmid by mixing both plasmids at equimolar concentrations.</li>
	<li>Electroporate the injected brain.
	<ol>
		<li>Place the head between the electrodes with the negative electrode positioned dorsally and parallel to the head and the positive electrode towards the ventral side of the head to target the MGE.</li>
		<li>Once the electrodes are well positioned, deliver 4 square pulses of 40 V for 50 ms at 500 ms inter-pulse intervals.</li>
		<li>Remove any residual tissue from the electrodes using tweezers already placed in the biosafety cabinet. 
		NOTE: These parameters have been optimized specifically for the electroporator used in our experiments. We recommend that the users perform optimizing tests beforehand if using a different type of electroporator.</li>
		<li>Repeat steps 4.1 to 4.3 for all remaining brains. 
		NOTE: Although this protocol describes the manipulations required for one brain, it is possible to inject up to 4 brains sequentially before electroporating each brain, thus increasing the yield. This strategy is especially advantageous when 2 or more different plasmids are injected sequentially (e.g. control or experimental plasmid) during the same experiment (allowing for comparison between littermates). In addition, it is possible to inject and electroporate simultaneously both sides of the brain to increase the yield, by positioning the electrodes completely parallel to the brain surface.</li>
	</ol>
	</li>
</ol>
5. Brain Dissection and Organotypic Slice Cultures
<ol>
	<li>While still manipulating in the sterile environment of the biosafety cabinet, dissect the brain out of the skull.
	<ol>
		<li>Stabilize the head on the layer of black wax by inserting a needle into each eye while carefully avoiding the brain.</li>
		<li>Use a pair of fine tweezers to hold the left side of the neck and a second pair of fine tweezers to tear the skin from the skull, from back to front.</li>
		<li>While holding the head laterally with tweezers in one hand, use another pair of tweezers in the other hand to carefully cut the skull at the level of the brainstem and gently pull the skull up. With each tweezer, cut the skull in the sagittal plane (midline) towards the front, and then incise laterally to liberate the skull fragments.</li>
		<li>Lift the brainstem and carefully cut the meninges and cranial nerves until the brain is completely out of the skull. 
		NOTE: All steps described in 5.1 should be performed under stringent sterile conditions in a biosafety cabinet.</li>
	</ol>
	</li>
	<li>Embed the brain in 4% low-melting point agarose for sectioning.
	<ol>
		<li>Fill a 35-mm petri dish with the agarose solution prepared above (kept liquid at 42 &#176;C).</li>
		<li>Quickly transfer an electroporated brain to the agarose-filled dish using the previously cut transfer pipette. Keep the dish at room temperature.</li>
		<li>Stir the agarose with a metal stick to keep the brain in the middle of the well (to prevent sinking) and position the brain in a rostro-caudal plane parallel to the dish. Stop stirring when the agarose starts to solidify, to avoid any brain damage.</li>
		<li>Use a razor blade to cut the agarose surrounding the brain in order to form a rectangular block, leaving a margin of 1 - 2 millimeters around the brain. Ensure that the rostral part of the brain is perpendicular to the anterior limit of the block to facilitate orientation for the subsequent sectioning steps.</li>
		<li>Repeat for each brain. 
		NOTE: It is possible to cut more than one brain at a time (maximum 3) by shaping separate agarose blocks while setting each brain at different heights.</li>
	</ol>
	</li>
	<li>Vibratome coronal sections and slice culture.
	<ol>
		<li>Thaw one aliquot of 100X N-2 supplement (150 &#181;L) on ice and add to 15 mL aliquoted culture medium under sterile conditions.</li>
		<li>Transfer 750 &#181;L of culture medium (with 1X N2 supplement) to each well of a 6-well culture plate.</li>
		<li>With curved tweezers, place one cell culture insert (30 mm diameter, 0.4 &#181;m pore size, PTFE) in each medium-filled well.</li>
		<li>Fill the vibratome bath with continuously oxygenated ACSF. Cool to 4 &#176;C with ice surrounding the bath, or use a refrigerated vibratome.</li>
		<li>Set the vibratome speed to 0.150 mm/s and the frequency to 80 Hertz.</li>
		<li>Glue the agarose block on the vibratome platform, rostral edge facing down and ventral edge facing the user.</li>
		<li>Cut the brain in coronal sections to obtain 250 &#181;m-thick sections (at 4 &#176;C).</li>
		<li>With sterilized spatulas, collect 2 - 3 sections containing the MGE and place all brain sections from one animal on a single 30-mm membrane insert, while carefully avoiding overlap between sections. Place the insert in one well of a 6-well culture plate (containing 750 &#181;L of supplemented culture medium, as described above). Alternatively, each section can be placed on a separate 13-mm diameter membrane in a 12-well culture plate filled with 500 &#181;l supplemented culture medium. The amount of culture medium recommended for each well allows the brain sections to be nourished by the media without being submerged. 
		NOTE: The steps described in 5.3.6 and 5.3.7 are not carried out under complete sterile conditions, unless the vibratome can be sterilized and used in a biosafety cabinet. Therefore, it is crucial to conduct these steps carefully to avoid any contamination. Appropriate protective equipment (clean mask, surgical gloves and lab coat) should be worn at all times and body parts, even covered, such as hair, face and hands, should never pass over culture plates (with or without culture medium). It is also recommended to spray 70% ethanol frequently on the gloves and spatulas used to collect the brain sections.</li>
		<li>Place the culture plate in a ventilated sterile incubator at 37 &#176;C with 60% humidity and 5% CO2 for 48 or 72 h. 
		NOTE: These incubation times were optimized for time-lapse imaging of MGE-derived INs and confocal imaging of fixed slices, respectively. Optimal incubation times should be tested beforehand for each experimental design. In addition, if the chosen incubation is 72 h and under, there is no need to change the culture medium. For longer incubation times, the culture medium should be changed every 2 - 3 days.</li>
		<li>After the desired incubation time, transfer the sections of interest to an 8-chambered coverslip and add 3 - 5 &#181;L of culture medium. Place the coverslip in an environmental chamber (37 &#176;C, 60% humidity, 5% CO2) connected to an inverted spinning disk confocal equipped with a computer-assisted acquisition software to set-up the time-lapse imaging session. 
		NOTE: Alternatively, sections can be fixed with 4% paraformaldehyde (overnight at 4 &#176;C or 2 h at room temperature) and subsequently immunostained with different antibodies for visualization of the morphological features of electroporated INs under a confocal microscope. Although eGFP and mCherry can be visualized by confocal microscopy without any counter-staining procedure, we recommend performing immunohistochemistry against GFP and mCherry to enhance the signal since the fixation process can reduce fluorescence, reducing the detection of finer components of embryonic neurons, such as smaller branches in the leading or trailing processes.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose. The views expressed herein do not necessarily represent the views of the Minister of Health or the Government of Canada.

In this article, we provide a reliable method for performing ex utero electroporation of the mouse MGE at e13.5 and for the generation of organotypic cultures of embryonic brain slices. Although in vitro methods, such as the Boyden Chamber Assay, are relatively easy to perform and can be used to assess the specific roles of different genes and proteins without the interference of other factors, they preclude the investigation of IN migration dynamics with regards to directionality and migration path<sup...

Cortical GABAergic interneurons (INs) are diverse with regards to their biochemical properties, physiological properties and connectivity, and they mediate different functions in mature networks1,2,3,4,5. The specification of different subtypes of cortical INs is tightly regulated through genetic cascades that have been extensively studied1,2. The majority (70%) of cortical GABAergic INs originate from progenitors in the medial ganglionic eminence (MGE), a ventrally located embryonic structure, and must migrate across relatively long distances to reach the cortical plate1,2,6. While cortical pyramidal cells migrate radially from the ventricular zone (VZ) to the cortical plate along the radial glia scaffold, the tangential migration of INs, which are not attached to such a scaffold, requires a variety of intrinsic and extrinsic cues to attract migrating neurons towards the cortical plate, while guiding them away from non-cortical structures2,7,8. After exiting the cell cycle, INs are repelled from the MGE by chemo-repulsive cues expressed within the VZ of the MGE, which triggers tangential migration towards the cortical plate9,10. Migrating INs avoid the striatum with the aid of different repulsive cues11 and, after reaching the cortical plate, they switch from a tangential to a radial migration mode and reach their final laminar position, partly in response to cues from pyramidal cells12 and other cellular populations13. The migration of INs, as for other neuronal populations, involves various dynamic morphological changes to permit the actual movement of the neuron. This so-called neuronal locomotion is characterized by repetitive cycles of three successive steps: the elongation of a leading process, an active anterograde motion of the nucleus (nucleokinesis), and the retraction of the trailing process14. IN migration is regulated by numerous intrinsic and extrinsic cues that drive the branching and active remodeling of the leading process to guide INs in the proper direction, determining both orientation and speed of migration14,15,16.
The determinants regulating cortical IN migration have been extensively studied in recent years1,2,7,17,18,19,20, and disruption in some of these molecular actors has been postulated to lead to neurodevelopmental disorders, such as pediatric refractory epilepsy or autism spectrum disorders1,2,21,22,23,24. Therefore, the development of various in vitro and in vivo approaches has been pursued to significantly advance our ability to study this dynamic process, as previously reviewed25. In vitro methods, including the Boyden chamber assay and the Stripe Choice Assay, provide the fastest and most reproducible means of assessing the requirement and cell-autonomous impact of specific genes or proteins during neuronal migration, without the influence of other factors25. These assays are particularly useful when combined with live-imaging8,26,27. With these techniques, INs are easily retrieved from e13.5 MGE and isolated by enzymatic and mechanical dissociation, after which different signaling pathways and guidance cues can be investigated, as illustrated previously8,28. However, these assays take place in an artificial extracellular matrix in the absence of three-dimensional tissue architecture, which may alter neuronal behavior and cell properties, potentially affecting cell migration and/or survival25. To circumvent these limitations, ex vivo MGE explants have been developed as an alternative tool to quantify the dynamic morphological changes occurring during migration along with parameters such as speed and orientation14,29. Generating MGE explants is relatively straightforward and has been extensively described elsewhere30. It entails the plating of a small extract of the MGE on a monolayer of mixed cortical cells or in a mixture of matrigel and collagen in the presence of attractive or repulsive cues25, although the latter are optional31. MGE explants allow for high resolution imaging of sparsely labeled cells, simplifying the study of intracellular processes, such as cytoskeletal remodeling during leading process branching, as shown previously32,33,34 and in the present study. MGE explants have been used successfully to assess dynamic cytoskeletal changes during IN migration in a 2D environment, for instance after specific pharmacological or chemotactic manipulations (see, for example, Tielens et al. 201633). However, with this approach, INs migrate within an artificial matrix, and this might alter IN behavior and the reproducibility and significance of the experimental results.
By contrast, in utero electroporation enables the genetic manipulation of INs in their native environment and is a widely used method to rapidly and efficiently assess the impact of gain and loss of gene function while circumventing the limitations of costly and time-consuming knockout and knock-in strategies25,35. In utero electroporation can be biased towards IN progenitors by using cell type specific promoters and by positioning the electrodes towards ventromedial structures, including the MGE36. Furthermore, in utero electroporation allows for the timely expression of experimental constructs within 1 - 2 days, as compared to the 7 - 10 days required for construct expression using viral vectors25. However, in utero electroporation of IN progenitors tends to be low-yield. Indeed, although pyramidal cell progenitors located in the dorsal ventricular zone can be efficiently transfected using in utero electroporation, targeting more ventrally located structures, such as the MGE, is more technically challenging, especially in small e13.5 embryos, and the high rate of embryonic lethality further reduces the experimental yield25.
To circumvent some of the technical limitations associated with in vitro MGE explant experiments and in vivo in utero electroporation, ex vivo organotypic slice cultures have been developed8,37,38,39. Brain organotypic slice cultures offer the advantage of mimicking in vivo conditions, while being less expensive and time-consuming than generating animal models25. Indeed, these preparations allow an easy access to the MGE, along with the specific visualization of INs, and can be combined with focal electroporation to investigate specific molecular pathways in INs migrating in a more physiological environment8,39,40,41. We have therefore optimized an approach for organotypic cultures38, which we combined with ex utero electroporation and time-lapse confocal imaging, to further assess the morphological and dynamic process occurring during tangential migration of MGE-INs. The present protocol was adapted and optimized from others who have used ex utero or in utero brain electroporation and organotypic slice cultures to study the migration of pyramidal cells42,43 and cortical INs36,39,44. Specifically, mouse embryos are decapitated and the MGE is electroporated ex vivo after the intraventricular injection of the experimental plasmids, allowing more efficient and precise targeting of MGE progenitors than what can be achieved with in utero electroporation. The brains are then extracted and sectioned into whole brain coronal slices that can be cultured for a few days, thus allowing continuous tracking and imaging of transfected INs. This approach typically labels 5 - 20 tangentially migrating INs per brain slice, minimizing the number of experimental iterations required to reach statistical significance, while labeling a sufficiently sparse neuronal population to ensure easy separation of individual neurons for reconstruction and fine morphological assessment. Furthermore, compared to MGE explants, organotypic cultures ensure that migrating INs are exposed to a more natural environment, including locally secreted chemokines and inputs from thalamic afferents. This approach is thus well suited to quantify the directionality and migratory path adopted by transfected INs, while offering sufficient anatomical details to allow the characterization of finer dynamic processes such as leading process branching, nucleokinesis and trailing process retraction.

<table><tbody><tr><td>Neurobasal Medium</td><td>ThermoFisher Scientific</td><td>21103049</td><td>Commercially available neuron-specific culture medium. Complete formulation available on this website: https://www.thermofisher.com/ca/en/home/technical-resources/media-formulation.251.html</td></tr><tr><td>B-27 serum-free supplement</td><td>ThermoFisher Scientific</td><td>17504044</td><td>50X Serum-free neuron specific supplement</td></tr><tr><td>15 mL sterile centrifuge tubes</td><td>Sarstedt</td><td>62.554.002</td><td></td></tr><tr><td>Leibovitz&#039;s (1X) L-15 Medium (+ L-Glutamine)</td><td>ThermoFisher Scientific</td><td>11415064</td><td>Commercially available neural-based culture medium supplemented with amino acids, vitamins and inorganic salts. Complete formulation available on the distributor&#039;s website&amp;nbsp;</td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030-081</td><td></td></tr><tr><td>Horse serum, heat inactivated</td><td>Millipore-Sigma</td><td>H1138-500ML</td><td></td></tr><tr><td>Neurocell supplement N-2 100X</td><td>Wisent</td><td>305-016</td><td>Botteinstein&#039;s N-2 Formulation</td></tr><tr><td>VWR Square PETG Media Bottles 125 mL</td><td>VWR</td><td>89132-062</td><td></td></tr><tr><td>Class II Type A Biosafety Cabinet</td><td>Nuaire</td><td>NU-540</td><td></td></tr><tr><td>Sucrose</td><td>BioShop</td><td>SUC700.1</td><td></td></tr><tr><td>Sodium Chloride</td><td>BioShop</td><td>SOD001.1</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>ThermoFisher Scientific</td><td>S233-500</td><td></td></tr><tr><td>D+ glucose</td><td>Millipore-Sigma</td><td>G7528-250G</td><td></td></tr><tr><td>Potassium Chloride</td><td>ThermoFisher Scientific</td><td>P217-500</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous</td><td>BioShop</td><td>SPM400.500</td><td></td></tr><tr><td>Calcium chloride dihydrate&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>BiosShop</td><td>MAG522</td><td></td></tr><tr><td>Agarose</td><td>BioShop</td><td>AGA002.500</td><td></td></tr><tr><td>50 mL sterile centrifuge tubes</td><td>Sarstedt</td><td>62.547.004</td><td></td></tr><tr><td>1.5 mL centrifuge tubes</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>P-97 Flaming/Brown Micropipette puller</td><td>Sutter Instruments Co.</td><td>Model P-97</td><td></td></tr><tr><td>0.4 mm I.D. x 75 mm Capillary Tube</td><td>Drummond scientific</td><td>1-000-800/12</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>E193</td><td></td></tr><tr><td>5 mL syringe</td><td>Becton Dickinson &amp;amp; Co</td><td>309646</td><td></td></tr><tr><td>Mineral Oil (heavy)</td><td>Rougier Pharma</td><td></td><td></td></tr><tr><td>WPI Swiss Tweezers #5</td><td>World Precision Instruments</td><td>504511</td><td>11 cm, straight, 0.06x0.07mm tips, antimagnetic. You will need 2 of these.</td></tr><tr><td>WPI Swiss Tweezers #7</td><td>World Precision Instruments</td><td>504504</td><td>11.5 cm, 0.18x0.2mm, curved tips</td></tr><tr><td>HTC Tweezers</td><td>World Precision Instruments</td><td>504617</td><td>11 cm, Straight, flat</td></tr><tr><td>Operating scissors</td><td>World Precision Instruments</td><td>501225</td><td>16 cm, Sharp/sharp, straight. You will need 3 of these.</td></tr><tr><td>Dressing Forceps</td><td>World Precision Instruments</td><td>501217</td><td>12.5 cm, straight, serrated</td></tr><tr><td>Iris Forceps</td><td>World Precision Instruments</td><td>504478</td><td>10.2 cm, full curve, serrated</td></tr><tr><td>DeBakey Tissue Forceps</td><td>World Precision Instruments</td><td>501996</td><td>15 cm, 45&amp;deg; angle, Delicate Jaw, 1.5mm wide</td></tr><tr><td>Fisherbran Microspatula with rounded ends</td><td>FisherScientific</td><td>21-401-5</td><td>You will need 2 of these.</td></tr><tr><td>Nanoject II Auto-Nanoliter Injector</td><td>Drummond scientific</td><td>3-000-204</td><td></td></tr><tr><td>TC Dish 60, Standard</td><td>Sarstedt</td><td>83.3901</td><td>60-mm dish</td></tr><tr><td>Tissue culture dish</td><td>Sarstedt</td><td>83.1800</td><td>35-mm dish</td></tr><tr><td>Black Wax</td><td>FisherScientific</td><td>S17432</td><td></td></tr><tr><td>Transfer pipettes&amp;nbsp;</td><td>Ultident</td><td>170-CTB700-212</td><td>3 mL, small bulb</td></tr><tr><td>Stereo Microscope</td><td>Leica Biosystems</td><td>Leica M80</td><td>In replacement to our stereomicroscope which has been discontinued by the manufacturer (StereoMaster from FisherScientific)</td></tr><tr><td>Electro Square Porator</td><td>BTX Harvard Apparatus</td><td>ECM 830</td><td></td></tr><tr><td>Tweezertrodes, Plattinum Plated, 3mm</td><td>BTX Harvard Apparatus</td><td>45-0487</td><td></td></tr><tr><td>25G 1 1/2</td><td>Becton Dickinson &amp;amp; Co</td><td>305127</td><td></td></tr><tr><td>Leica VT1000S Vibrating blade microtome</td><td>Leica Biosystems</td><td>VT1000S</td><td></td></tr><tr><td>GEM, Single edge razor blade</td><td>Electron Microscopy Sciences</td><td>71952-10</td><td>Remove the blunt end before inserting in the blade designated space of the vibratome</td></tr><tr><td>&amp;micro;-Slide 8 well</td><td>Ibidi</td><td>80827</td><td>Pack of 15</td></tr><tr><td>Millicell cell culture insert</td><td>Millipore-Sigma</td><td>PICM0RG50</td><td>30 mm, hydrophilic PTFE, 0.4 &amp;micro;m pore, pack of 50.&amp;nbsp;</td></tr><tr><td>Leica DMi6000 microscope</td><td>Leica Microsystems</td><td>N/A</td><td></td></tr><tr><td>Spinning disk confocal head Ultraview Vox</td><td>Perkin Elmer</td><td>N/A</td><td></td></tr><tr><td>Volocity 6.0 acquisition software</td><td>Improvision/Perkin Elmer</td><td>N/A</td><td></td></tr><tr><td>LiveCell Stage top incubation system</td><td>Pathology devices</td><td>LC30030</td><td>Provides Temperature, CO2 and humidity control.&amp;nbsp;</td></tr><tr><td>SP8 confocal microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>mCherry-Lifeact-7</td><td>Addgene</td><td>54491</td><td>Gift from Michael Davidson</td></tr><tr><td>Fast Green FCF</td><td>Millipore-Sigma</td><td>F7258-25G</td><td>25G bottle, certified by the Biological Stain Commission</td></tr></tbody></table>

ex utero electroporation and organotypic slice cultures of embryonic mouse brains for live-imaging of migrating gabaergic interneurons

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

In this section, we provide representative results obtained following the ex utero electroporation of a control plasmid, or an experimental plasmid targeting a gene of interest, in the MGE of e13.5 mouse embryos followed by organotypic slice cultures incubated at 37 °C for 48 h (for time-lapse imaging) or 72 h (for fixation and immunohistochemical labeling) (see Figure 1B for schematic protocol). Representative examples of INs migrating from an MGE e...

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

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Research

JoVE Journal

Neuroscience

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Source: Wiegreffe, C., et. al.,  Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation. J. Vis. Exp. (2017) This video demonstrates time-lapse confocal microscopy to observe cortical neuron radial migration from the subventricular zone to the cortical plate via radial glial scaffolds in transduced mouse embryonic brain slices.

Time-Lapse Imaging of Cortical Neuron Radial Migration in Transduced Mouse Embryonic 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.

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

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions.  Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotypic Hippocampal Slice Cultures

Learn ex utero electroporation and organotypic slice cultures to visualize migrating GABAergic interneurons in embryonic mouse brains. This low-cost method enables live-imaging of neuronal development for neurodevelopmental research.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons (Video) | JoVE Journal

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

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