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>
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			<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>
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			<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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			<td>15 mL sterile centrifuge tubes</td>
			<td>Sarstedt</td>
			<td>62.554.002</td>
			<td></td>
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			<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>
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			<td>Horse serum, heat inactivated</td>
			<td>Millipore-Sigma</td>
			<td>H1138-500ML</td>
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			<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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			<td>VWR Square PETG Media Bottles 125 mL</td>
			<td>VWR</td>
			<td>89132-062</td>
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			<td>Class II Type A Biosafety Cabinet</td>
			<td>Nuaire</td>
			<td>NU-540</td>
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			<td>Sucrose</td>
			<td>BioShop</td>
			<td>SUC700.1</td>
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			<td>Sodium Chloride</td>
			<td>BioShop</td>
			<td>SOD001.1</td>
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			<td>ThermoFisher Scientific</td>
			<td>S233-500</td>
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			<td>D+ glucose</td>
			<td>Millipore-Sigma</td>
			<td>G7528-250G</td>
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			<td>Potassium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>P217-500</td>
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			<td>Sodium phosphate monobasic anhydrous</td>
			<td>BioShop</td>
			<td>SPM400.500</td>
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			<td>Calcium chloride dihydrate&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>C79-500</td>
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			<td>Magnesium sulfate heptahydrate</td>
			<td>BiosShop</td>
			<td>MAG522</td>
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			<td>Agarose</td>
			<td>BioShop</td>
			<td>AGA002.500</td>
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			<td>50 mL sterile centrifuge tubes</td>
			<td>Sarstedt</td>
			<td>62.547.004</td>
			<td></td>
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		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
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		<tr>
			<td>P-97 Flaming/Brown Micropipette puller</td>
			<td>Sutter Instruments Co.</td>
			<td>Model P-97</td>
			<td></td>
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		<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>
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		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>E193</td>
			<td></td>
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		<tr>
			<td>5 mL syringe</td>
			<td>Becton Dickinson &#38; Co</td>
			<td>309646</td>
			<td></td>
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		<tr>
			<td>Mineral Oil (heavy)</td>
			<td>Rougier Pharma</td>
			<td></td>
			<td></td>
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			<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>
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		<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>
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		<tr>
			<td>Nanoject II Auto-Nanoliter Injector</td>
			<td>Drummond scientific</td>
			<td>3-000-204</td>
			<td></td>
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			<td>TC Dish 60, Standard</td>
			<td>Sarstedt</td>
			<td>83.3901</td>
			<td>60-mm dish</td>
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			<td>Tissue culture dish</td>
			<td>Sarstedt</td>
			<td>83.1800</td>
			<td>35-mm dish</td>
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			<td>Black Wax</td>
			<td>FisherScientific</td>
			<td>S17432</td>
			<td></td>
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			<td>Transfer pipettes&#160;</td>
			<td>Ultident</td>
			<td>170-CTB700-212</td>
			<td>3 mL, small bulb</td>
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			<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>
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			<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>
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		<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 ...

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