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

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 explant are also illustrated (see Figure 1C for schematic protocol) as a comparison to the method described here. The electroporation of a plasmid in MGE progenitors seems to delay the exit of INs from the MGE and culture time must be adjusted accordingly.

In a successful experiment, organotypic slices appear healthy under the confocal microscope, i.e. cortical layers are easily distinguishable using the bright field mode and there is no visible contamination on the slice surface (recognizable by intense autofluorescence and the presence of fluorescent filaments visible under epifluorescence). In healthy chronic organotypic slices, approximately 20% of cells undergo apoptosis after 72 h in culture (Figure 2), as previously described in chronic organotypic cultures (e.g. postnatal cultures)⁵². Nonetheless, gross cytoarchitectural brain structure remains well defined, as illustrated here using DAPI staining (Figure 2A). Our protocol for ex vivo electroporation of the MGE yields an average of 50 – 100 transfected cells per slice (Figure 3A,B), of which 5 – 20 cells will be seen migrating dorsally after 72 h in culture. After fixation and immunohistochemical staining, INs migrating tangentially towards the cortical plate can be easily identified with confocal microscopy as they present an elongated/oval cell body, the occasional trailing process, and a leading process oriented tangentially. The leading process usually gives rise to one or two branches and is recognizable by the presence of an occasional swelling in front of the nucleus (housing the centrosome before nucleokinesis) and growth cones at the end of each branch. (Figure 3C-G).

This protocol was designed for the observation of migrating MGE-derived INs at the single-cell level using time-lapse imaging. For this particular experiment (Figure 4), coronal organotypic brain slices were generated from Dlx5/6^Cre;RCE^EGFP embryos electroporated at e13.5 with a plasmid expressing an experimental shRNA (targeting a gene of interest) and the TdTomato cassette. The slices were incubated for 48 h, transferred to an 8-well chambered coverslip dish (1 slice per chamber floating on a thin layer of supplemented culture medium) and imaged every 3 minutes for 6 – 8 h using a 20x air (0.70 NA) objective on an inverted microscope equipped with a spinning disk confocal head, a computer-assisted acquisition software and a stage-top environmental chamber. During imaging, the slices were kept at 37 °C and were continuously oxygenated and humidified in the environmental chamber (5% CO₂ and 60% H₂O). Electroporated MGE-derived INs were identified as such by their expression of eGFP, confirming their GABAergic IN identity, and their expression of TdTomato, confirming that they express the experimental plasmid (Figure 4A,B). Electroporated INs are easily identifiable and well isolated, as seen in Figure 4, allowing for the finer analysis of migration dynamic parameters, such as speed and distance traveled. In addition, labeled INs are seen migrating tangentially towards the cortical plate, while in their natural environment, enabling us to identify dynamic morphological changes such as branching of the leading process and nucleokinesis (Figure 4C-F).

The ex vivo electroporation of MGE-derived INs can also be combined with MGE explants to enable high resolution imaging of dynamic cytoskeletal processes occurring during migration, as a complement to the study of directionality and migratory dynamics in organotypic cultures. As an example, different phases of neuronal locomotion reminiscent of those occurring during tangential migration of INs in organotypic slice cultures can be observed in electroporated INs migrating from an MGE explant 48 h after electroporation, such as leading neurite extension and nucleokinesis (Figure 5B-E). Various cytoskeletal processes can then be studied at high resolution in isolated cells migrating from an MGE explant, for instance by staining F-actin structures with phalloidin (Figure 6A), which cannot be achieved optimally in organotypic slices given the high cellular density (and thus of cytoskeletal elements) in a native environment. These cytoskeletal processes, and in particular F-actin remodeling, can further be studied dynamically by combining Lifeact expression with time-lapse imaging. For instance, we show an example of high-resolution time-lapse imaging of an IN derived from an MGE explant obtained from a Nkx2.1^Cre;RCE^EGFP mouse brain carrying a targeted deletion of a gene of interest in INs. This IN was transfected with the mCherry-Lifeact-7 plasmid under the control of the CMV promoter (gift from Michael Davidson), using the method schematized in Figure 1C, allowing for the tracking of F-actin remodeling occurring in real-time (Figure 6B). Thus, while organotypic cultures are required to properly assess directionality or migration path of genetically modified INs, MGE explants can complement such studies by providing better access to isolated cells for high-resolution imaging of dynamic cytoskeletal processes.

Technical pitfalls may result in failure of the experiments described above. For instance, embedding the brains in an agarose solution warmer than 42 °C can result in tissue destruction and neuronal death, revealed by a loss of translucency of the brain or slices, which become opaque. However, the temperature should not be kept too low, as a solidifying agarose solution can destroy the fragile embryonic brain during the embedding process. Secondly, contamination can significantly reduce the yield of the experimental approaches described here. Thus, all steps should be carried out with care, under stringent sterile conditions as much as feasible. All solutions and equipment should be sterilized before use and equipment should be frequently sprayed with 70% ethanol to avoid contamination from either bacteria or mold. Contamination can be visible directly in the culture wells, as the culture medium becomes yellow or opaque. Contamination may go unnoticed when the culture medium remains clear and fluid. However, a layer of mold on the slice surface can usually be observed or the slices become excessively fragile during the fixation and staining steps. When such slices are visualized under the microscope, contamination may manifest as a layer of autofluorescence over labeled neurons or be revealed by the presence of long fluorescent filaments. Organotypic slices kept in contaminated wells should be thrown out, but the slices cultured in the other wells of the same plate can be kept for further steps. Bacterial contamination is quite rare but should be taken seriously, meaning that all equipment, including shared incubators, and culture room should be manually cleaned and sterilized.

Figure 1: Schematic representations of the protocols for ex utero electroporation followed by organotypic slice culture or MGE explants. A. Example of a biosafety cabinet setup with all sterilized instruments needed for the protocol described here. B. Schematic representation of the protocol used for the ex utero electroporation and organotypic slice cultures. Briefly, the embryos are decapitated, the experimental plasmid is injected in the lateral ventricle (1) and electroporated in the MGE (2). The brain is microdissected out of the skull (3) and embedded in agarose (4). Organotypic sections are generated on a vibratome (5) and placed in culture at 37 °C(6) for 48 h for time-lapse microscopy (7.1) or 72 h for cell reconstructions (7.2). C. Schematic representation of the protocol adapted from Myers et al. 2013⁵³ and used for the generation of MGE explants. Briefly, dorsolateral cortices are first dissected out from e12.5 to e14.5 wild-type mouse embryos (1) and dissociated mechanically in supplemented culture medium (2). Cortical cells are plated at a density of 5.25 x 10⁵ cortical cells/cm² and cultured for 2 h at 37 °C in a collagen/poly-L-lysine-coated 8-chambered coverslip (3). Then, e13.5 Dlx5/6^Cre;RCE^EGFP embryos are processed for ex utero electroporation of the MGE (4, 5), and the MGE is manually isolated from the brain and cut in 100 µm² fragments (6). These explants are then placed on top of the previously prepared cortical feeder layer, covered with culture medium and co-cultured for 48 h before time-lapse imaging (7). Please click here to view a larger version of this figure.

Figure 2: Preserved cytoarchitecture in healthy organotypic slices. The generation of organotypic slice cultures after ex vivo electroporation of Nkx2.1^Cre;RCE^EGFP mouse embryos (A) does not result in significant tissue damage, as revealed by preserved cytoarchitecture (DAPI (blue) and Cre-reporter (GFP)), when compared to an 18 µm-thick cryosection from a Nkx2.1^Cre;RCE^EGFP embryo transcardially perfused with 4% PFA at e15.5. D and M indicate dorso-ventral and latero-medial axes, respectively. (B). High magnification images taken with an inverted confocal microscope equipped with a 63x/1.4 oil objective after staining with an anti-Cleaved-Caspase 3 antibody (Cl-cas3) reveal that approximately 20% of the cells in the cortical plate undergo apoptosis (white arrowheads) after 72 h in culture, while GFP-positive INs stay healthy (white arrow), as exemplified in the photomicrographs in C-C'''. By comparison, cellular apoptosis is almost completely absent from the thin cryosection from a perfused animal (D-D'''). Scale bars: 250 µm (A-B) and 15 µm (C-D'''). Please click here to view a larger version of this figure.

Figure 3: Organotypic slices of mice brain embryos and high magnification photomicrographs of electroporated interneurons (INs). A. An organotypic slice culture from a Dlx5/6^Cre;RCE^EGFP mouse brain (in which all INs are green) electroporated with a Dlx5/6::shRNA^scrambled-IRES-TdTomato plasmid. The slice was fixed with 4% PFA after 72 h in culture, and immunostained for GFP and mCherry. Electroporated INs were imaged in 3D (50 – 60 µm-thick z-stacks with optical sections taken every 0.5 µm) using a confocal microscope equipped with a 63x/1.3 NA oil objective. INs migrating tangentially in the sub-ventricular zone of the dorsal pallium are readily identifiable (red, open arrow). D and M indicate dorso-ventral and latero-medial axes, respectively. B. A representative organotypic slice culture from a wildtype (WT) embryo electoporated with a control Dlx5/6::shRNA^scrambled-IRES-TdTomato plasmid, fixed at 72 h and immunostained for mCherry only. Electroporated INs (red) are seen migrating dorsally towards the cortical plate. Electroporated INs are identified by their co-expression of TdTomato and EGFP in Dlx5/6^Cre;RCE^EGFP mice (C-D), or by their expression of TdTomato only in WT mice (E-H), and by their typical morphology, i.e. an oval cell body, a branched leading process (white arrowheads, D-H), an occasional trailing process (white arrow, C-D) and an occasional swelling of the leading process housing the centrosome before nucleokinesis (white star in C-C' and G). Scale bars: 250 µm (A-B) and 25 µm (C-H). Please click here to view a larger version of this figure.

Figure 4: Time-lapse live-imaging of electroporated INs in organotypic slices cultured for 48 h. INs electroporated at e13.5 with an experimental plasmid expressing the TdTomato cassette and a shRNA targeting a gene of interest are seen in tangential migration in an organotypic brain slice obtained from a Dlx5/6^Cre;RCE^EGFP mouse embryo after 48 h of culture (A, white square). In B, the same electroporated IN (white square) is seen in the process of nucleokinesis, while migrating tangentially in the cortex, i.e. parallel to the pial surface,and is followed in time-lapse imaging every 3 minutes for 8 h using a 20x/0.85 NA air objective (enlarged boxes, C-F). The neuron initially displays an elongated cell body (open arrow) in the process of nucleokinesis (C), as well as a trailing process (white arrow) and a branched leading process (white arrowheads). After 3 h of live-imaging, the nucleokinesis is completed, the trailing process has retracted, and the leading process is extending two long branches (D, white arrowheads). After 5 h 30 min, one of the leading process branch has retracted (white arrowheads) and the neuron cell body (open arrow) has moved forward about 10 µm (E). Finally, after 8 h of imaging, migration has paused, the neuron has extended its leading process again and a trailing process has appeared (white arrow), but the nucleus (open arrow) has not yet moved forward (F). Scale bars: 250 µm (A), 70 µm (B) and 30 µm (C-F). Please click here to view a larger version of this figure.

Figure 5: Time-lapse imaging of INs derived from an MGE explant cultured for 48 h. INs are seen migrating out of an MGE explant obtained from a Nkx2.1^Cre;RCE^EGFP mouse (in which all MGE-derived INs express eGFP) electroporated with the CMV::mCherry-LifeAct-7 plasmid at e13.5, using the method adapted from Myers et al. 2013⁵³ and schematized in Figure 1C, and cultured for 48 h (A). INs were visualized using time-lapse live-imaging for 5 h (B-E). In this example, one IN co-expressing eGFP and LifeAct is seen initially in the process of nucleokinesis (open arrow) and extends two leading process branches (white arrowheads; B). This IN completes nucleokinesis after 1 h (see open arrow), as the cell body has moved forward and a trailing process has appeared at the rear of the cell body (white arrow), and still displays one leading process with two branches (white arrowheads; C). A second nucleokinesis is under way after 2 h 30 min (open arrows) and the IN is now endowed with two leading processes originating from the cell body and a longer trailing process (white arrow; D). Finally, after 5 h, the IN retracts one neurite while translocating the centrosome in the remaining leading process branch (white arrowhead) in preparation for a third nucleokinesis, with a trailing process (white arrow) still present at the rear of the cell body (E). Scale bars: 70 µm (A) and 20 µm (B-E). Please click here to view a larger version of this figure.

Figure 6: High-resolution time-lapse imaging of actin remodeling in INs from an MGE explant cultured for 48 h. A. An organotypic slice from a Nkx2.1^Cre;RCE^EGFP mouse brain is fixed at e15.5, stained with Alexa-594 Phalloidin, allowing for the visualization of filamentous actin. INs are imaged using a 63x/1.3 NA oil objective on a confocal microscope. In healthy INs, filamentous actin is seen cupping the rear of the cell body following the retraction of the trailing process after completion of nucleokinesis (white arrow, A'-A'''). B. An MGE explant is obtained as above from a Nkx2.1^Cre;RCE^EGFP mouse brain carrying a targeted deletion of a gene of interest and electroporated with a plasmid expressing mCherry-Lifeact-7 under the control of the CMV promoter. Time-lapse live imaging is performed after 48 h of culture, and electroporated INs are followed every 3 minutes for 3 h using a 40x/0.85 air objective. Active remodeling of the actin cytoskeleton occurs in the IN transfected with LifeAct, as exemplified by the movement of red (white in the black and white images) fluorescent dots within the leading process and growth cones (white arrowheads) and at the rear of the cell body during nucleokinesis (B-B''', white arrows). Scale bars: 7 µm (A-A''') and 10 µm (B-B'''). Please click here to view a larger version of this figure.