Name: ex utero electroporation and organotypic slice cultures of embryonic mouse brains for live-imaging of migrating gabaergic interneurons
Uploaded: 2018-04-20T15:00:00
Description: Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons at JoVE.com

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 C...

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>
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		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>Sarstedt</td>
			<td>62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;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&#39;s website&#160;</td>
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			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td></td>
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		<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&#39;s N-2 Formulation</td>
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		<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>
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		<tr>
			<td>Sodium Chloride</td>
			<td>BioShop</td>
			<td>SOD001.1</td>
			<td></td>
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		<tr>
			<td>Sodium bicarbonate</td>
			<td>ThermoFisher Scientific</td>
			<td>S233-500</td>
			<td></td>
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		<tr>
			<td>D+ glucose</td>
			<td>Millipore-Sigma</td>
			<td>G7528-250G</td>
			<td></td>
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		<tr>
			<td>Potassium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>P217-500</td>
			<td></td>
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		<tr>
			<td>Sodium phosphate monobasic anhydrous</td>
			<td>BioShop</td>
			<td>SPM400.500</td>
			<td></td>
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		<tr>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>C79-500</td>
			<td></td>
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		<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 &#38; 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&#176; 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&#160;</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 &#38; 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>&#181;-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 &#181;m pore, pack of 50.&#160;</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.&#160;</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

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 &#176;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 ...

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons at JoVE.com

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

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.

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 ...

The overall goal of this experiment is to visualize genetically modified cortical GABAergic interneurons during tangential migration in the most naturalistic environment possible. This method can help answer key questions in developmental neurosciences by screening the impact of the loss of function of specific genes of proteins during brain development. The main advantage of this technique is that it is low cost and relatively easy to perform compared to other similar methods such as in utero electroporation.So this technique can ultimately improve diagnosis for patients with genetic neurodevelopmental disorders such as autism and epilepsy by clarifying the pathogenecity of specific mutations identified in patients as well as the roles of novel disease genes during brain development. Begin by setting up the biosafety cabinet with all the instruments needed for dissection and culture. Generously spray all instruments in the biosafety cabinet with 70%ethanol and sterilize the instruments and cabinet interior with UV light for 15 to 20 minutes.When ready to collect embryos, generously spray the abdomen of the euthanized dam 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. With a second pair of sterilized forceps and scissors, pull the abdominal fascia up and cut it while carefully avoiding the uterus.Using a third pair of sterilized forceps and scissors, hold the uterine horns and cut them out of the pelvic cavity. Place the dissected uterine horns in a sterile 60 millimeter petridish filled with neural based lebovitz culture medium supplemented with amino acids, vitamins and inorganic salts. In the sterile biosafety cabinet, use two pairs of fine tweezers to dissect the embryos out of the placenta and isolate the heads by decapitation.Bevel cut the tip of a sterile three milliliter plastic transfer pipette and use this to aspirate the heads and transfer them into a new sterile 60 millimeter petri dish filled with the same supplemented neural based culture medium to wash out the blood. Transfer them again into a third petri dish layered with solidified black wax and filled with neural based supplemented culture medium. Place the 16 millimeter petri dish layered with black wax and containing the decapitated heads in neural based supplemented culture medium under the dissecting microscope in the biosafety cabinet.Use fine tweezers held in the left hand to stabilize the head with the rostral part facing right. Then use the nano injector in the right hand to inject one to two microliters of the dye spiked plasmid solution into the right lateral ventricle. Electroporate the injected brain by placing the head between the electrodes with the negative electrode positioned dorsally and parallel to the head and the positive electrode toward the ventral side of the head to target the MGE.Once the electrodes are well positioned, deliver foursquare pulses of 40 volts for 50 milliseconds at 500 millisecond interpulse intervals. While still working in the sterile environment of the biosafety cabinet, start dissecting the brain out of the skull. First, stabilize the head on the layer of black wax by inserting a needle into each eye while carefully avoiding the brain.Then 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. 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 pull the skull up. With each tweezer, cut the skull in the sagittal plane towards the front and then incise laterally to liberate the skull fragments.Then lift the brainstem and carefully cut the meninges and cranial nerves until the brain is completely out of the skull. Fill a 35 millimeter petri dish with molten agarose solution kept liquid at 42 degrees celsius. Then quickly transfer an electroporated brain to the agarose filled dish using the previously cut transfer pipette.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 rostral caudal plane parallel to the dish. Stop stirring when the agarose starts to solidify to avoid any damage to the brain. While waiting for the agarose embedding to solidify, transfer 750 microliters of N2 supplemented culture medium in each well of the six well culture plate according to the number of brains to process.And place a 30 millimeter membrane insert in each medium filled well. Use a razor blade to cut the agarose surrounding the brain to form a rectangular block leaving a margin of one to two 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.Glue the agarose block on the vibratome platform rostral edge facing down and ventral edge facing the user. Cut the brain in coronal sections to obtain 250 micron thick sections at four degrees celsius. With sterilized spatulas, collect two to three sections containing the MGE and place all brain sections from one animal on a single 30 millimeter membrane insert.While carefully avoiding overlap between sections. Place the culture plate in a ventilated sterile incubator at 37 degrees celsius with 60%humidity and five percent CO2 for 48 or 72 hours. After the desired incubation time, transfer the sections of interest to an eight chambered coverslip filled with three to five microliters of culture medium.Place the coverslip in an environmental chamber connect it to an inverted spinning disk confocal equipped with a computer-assisted acquisition software to set up the time-lapse imaging session. Interneurons electroporated at E 13.5 with an experimental plasmid expressing the TD tomato cassette and and SH RNA targeting a gene of interest are seen in tangential migration in an organo-typic brain slice obtained from a DLX5 6Cre RCEE GFP mouse embryo after 48 hours of culture. Interneurons were visualized using time-lapse live imaging for eight hours.In this example, the same electroporated interneuron is seen in the process of nucleokinesis. While migrating tangentially in the cortex parallel to the peel surface. The neuron initially displays an elongated cell body in the process of nucleokinesis.As well as a trailing process and a branched leading process. After three hours of live imaging, the nucleokinesis is completed. The trailing process has retracted and the leading process is extending two long branches.After five hours and 30 minutes, one of the leading process branches has retracted and the neuron cell body has moved forward about 10 microns. Finally, after eight hours of imaging, migration has paused, the neuron has extended its leading process again and a trailing process has appeared. But the nucleus has not yet moved forward.Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it's important to keep a sterile environment at all possible times. So following this procedures, other methods like MG electroporations followed by explants can be performed to address additional questions for answers to elucidate the dynamic cytoskeletal processes occurring during neuronal migration at the ultracellular level.

Research

JoVE Journal

Neuroscience

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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 ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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

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

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...