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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

All experiments involving animals were approved by the Comité 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'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

  1. Prepare 125 mL of sterile culture medium.
    1. 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 °C.
      NOTE: Prepared culture medium can be stored for up to 3 weeks at 4 °C.
    2. Divide a 100X stock solution of Botteinstein's N-2 formulation48 into 150 µL aliquots under sterile conditions and freeze at -20°C until use.
  2. Prepare 1 L of sterile artificial cerebrospinal fluid (ACSF).
    1. 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.
    2. Using a 0.22 µm filter, filter the solution into a sterile bottle in a sterilized biosafety cabinet and store at 4 °C for up to 1 month.
  3. Prepare a fresh solution of 4% agarose in ACSF before each experiment.
    1. Measure 25 mL of previously prepared ACSF in a 50-mL sterile conical tube and add 1 g of low-melting point agarose.
    2. 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 °C during the remainder of the experiment to avoid solidification.
      NOTE: Higher temperatures will damage brain tissues.

2. Preparation of Plasmids for Injection

  1. Pull a glass microinjection pipette
    1. 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.
    2. Press the pull button.
    3. 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.
  2. 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.
  3. During the sterilization step, thaw plasmids on ice (4 °C).
  4. Measure 10 µL of the plasmid from a stock solution (4 µg/µL) 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'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.
  5. After sterilization, prepare the nano-injector as follows.
    1. 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 µm.
      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.
    2. Use a syringe to fill the micropipette with mineral oil from its unpulled end (to expel all the air).
    3. Insert the filled glass micropipette in the nano-injector by following the manufacturer's instructions.
    4. Empty 2/3rds of the glass micropipette (keeping enough oil to prevent air entry).
  6. Carefully insert the prepared micropipette in the tube containing the plasmid/dye solution and fill the glass micropipette with the plasmid/dye solution.

3. Collection of Mouse Embryos from Pregnant Females

  1. 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).
  2. 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.
  3. Collect embryos by C-section as follows.
    1. 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.
    2. With a second pair of sterilized forceps and scissors, pull the abdominal fascia up and cut it while carefully avoiding the uterus.
    3. 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).
  4. 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.
  5. 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.

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.

  1. 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.
  2. 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 µL 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.
  3. Electroporate the injected brain.
    1. 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.
    2. Once the electrodes are well positioned, deliver 4 square pulses of 40 V for 50 ms at 500 ms inter-pulse intervals.
    3. 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.
    4. 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.

5. Brain Dissection and Organotypic Slice Cultures

  1. While still manipulating in the sterile environment of the biosafety cabinet, dissect the brain out of the skull.
    1. Stabilize the head on the layer of black wax by inserting a needle into each eye while carefully avoiding the brain.
    2. 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.
    3. 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.
    4. 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.
  2. Embed the brain in 4% low-melting point agarose for sectioning.
    1. Fill a 35-mm petri dish with the agarose solution prepared above (kept liquid at 42 °C).
    2. Quickly transfer an electroporated brain to the agarose-filled dish using the previously cut transfer pipette. Keep the dish at room temperature.
    3. 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.
    4. 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.
    5. 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.
  3. Vibratome coronal sections and slice culture.
    1. Thaw one aliquot of 100X N-2 supplement (150 µL) on ice and add to 15 mL aliquoted culture medium under sterile conditions.
    2. Transfer 750 µL of culture medium (with 1X N2 supplement) to each well of a 6-well culture plate.
    3. With curved tweezers, place one cell culture insert (30 mm diameter, 0.4 µm pore size, PTFE) in each medium-filled well.
    4. Fill the vibratome bath with continuously oxygenated ACSF. Cool to 4 °C with ice surrounding the bath, or use a refrigerated vibratome.
    5. Set the vibratome speed to 0.150 mm/s and the frequency to 80 Hertz.
    6. Glue the agarose block on the vibratome platform, rostral edge facing down and ventral edge facing the user.
    7. Cut the brain in coronal sections to obtain 250 µm-thick sections (at 4 °C).
    8. 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 µL 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 µl 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.
    9. Place the culture plate in a ventilated sterile incubator at 37 °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.
    10. After the desired incubation time, transfer the sections of interest to an 8-chambered coverslip and add 3 - 5 µL of culture medium. Place the coverslip in an environmental chamber (37 °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 °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.

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Results

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 ...

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Discussion

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

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Disclosures

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.

Acknowledgements

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 Québec-Santé (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.

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Materials

NameCompanyCatalog NumberComments
Neurobasal MediumThermoFisher Scientific21103049Commercially available neuron-specific culture medium. Complete formulation available on this website: https://www.thermofisher.com/ca/en/home/technical-resources/media-formulation.251.html
B-27 serum-free supplementThermoFisher Scientific1750404450X Serum-free neuron specific supplement
15 mL sterile centrifuge tubesSarstedt62.554.002
Leibovitz's (1X) L-15 Medium (+ L-Glutamine)ThermoFisher Scientific11415064Commercially available neural-based culture medium supplemented with amino acids, vitamins and inorganic salts. Complete formulation available on the distributor's website 
L-GlutamineInvitrogen25030-081
Horse serum, heat inactivatedMillipore-SigmaH1138-500ML
Neurocell supplement N-2 100XWisent305-016Botteinstein's N-2 Formulation
VWR Square PETG Media Bottles 125 mLVWR89132-062
Class II Type A Biosafety CabinetNuaireNU-540
SucroseBioShopSUC700.1
Sodium ChlorideBioShopSOD001.1
Sodium bicarbonateThermoFisher ScientificS233-500
D+ glucoseMillipore-SigmaG7528-250G
Potassium ChlorideThermoFisher ScientificP217-500
Sodium phosphate monobasic anhydrousBioShopSPM400.500
Calcium chloride dihydrate ThermoFisher ScientificC79-500
Magnesium sulfate heptahydrateBiosShopMAG522
AgaroseBioShopAGA002.500
50 mL sterile centrifuge tubesSarstedt62.547.004
1.5 mL centrifuge tubesSarstedt72.690.001
P-97 Flaming/Brown Micropipette pullerSutter Instruments Co.Model P-97
0.4 mm I.D. x 75 mm Capillary TubeDrummond scientific1-000-800/12
EthanolVWRE193
5 mL syringeBecton Dickinson & Co309646
Mineral Oil (heavy)Rougier Pharma
WPI Swiss Tweezers #5World Precision Instruments50451111 cm, straight, 0.06x0.07mm tips, antimagnetic. You will need 2 of these.
WPI Swiss Tweezers #7World Precision Instruments50450411.5 cm, 0.18x0.2mm, curved tips
HTC TweezersWorld Precision Instruments50461711 cm, Straight, flat
Operating scissorsWorld Precision Instruments50122516 cm, Sharp/sharp, straight. You will need 3 of these.
Dressing ForcepsWorld Precision Instruments50121712.5 cm, straight, serrated
Iris ForcepsWorld Precision Instruments50447810.2 cm, full curve, serrated
DeBakey Tissue ForcepsWorld Precision Instruments50199615 cm, 45° angle, Delicate Jaw, 1.5mm wide
Fisherbran Microspatula with rounded endsFisherScientific21-401-5You will need 2 of these.
Nanoject II Auto-Nanoliter InjectorDrummond scientific3-000-204
TC Dish 60, StandardSarstedt83.390160-mm dish
Tissue culture dishSarstedt83.180035-mm dish
Black WaxFisherScientificS17432
Transfer pipettes Ultident170-CTB700-2123 mL, small bulb
Stereo MicroscopeLeica BiosystemsLeica M80In replacement to our stereomicroscope which has been discontinued by the manufacturer (StereoMaster from FisherScientific)
Electro Square PoratorBTX Harvard ApparatusECM 830
Tweezertrodes, Plattinum Plated, 3mmBTX Harvard Apparatus45-0487
25G 1 1/2Becton Dickinson & Co305127
Leica VT1000S Vibrating blade microtomeLeica BiosystemsVT1000S
GEM, Single edge razor bladeElectron Microscopy Sciences71952-10Remove the blunt end before inserting in the blade designated space of the vibratome
µ-Slide 8 wellIbidi80827Pack of 15
Millicell cell culture insertMillipore-SigmaPICM0RG5030 mm, hydrophilic PTFE, 0.4 µm pore, pack of 50. 
Leica DMi6000 microscopeLeica MicrosystemsN/A
Spinning disk confocal head Ultraview VoxPerkin ElmerN/A
Volocity 6.0 acquisition softwareImprovision/Perkin ElmerN/A
LiveCell Stage top incubation systemPathology devicesLC30030Provides Temperature, CO2 and humidity control. 
SP8 confocal microscopeLeica
mCherry-Lifeact-7Addgene54491Gift from Michael Davidson
Fast Green FCFMillipore-SigmaF7258-25G25G bottle, certified by the Biological Stain Commission

References

  1. Rossignol, E. Genetics and function of neocortical GABAergic interneurons in neurodevelopmental disorders. Neural Plast. , 649325(2011).
  2. Jiang, X., Lachance, M., Rossignol, E. Involvement of cortical fast-spiking parvalbumin-positive basket cells in epilepsy. Prog Brain Res. 226, 81-126 (2016).
  3. Ascoli, G. A., et al. Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Nature Reviews Neuroscience. 9, 557-568 (2008).
  4. Klausberger, T., Somogyi, P. Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science. 321, 53-57 (2008).
  5. Somogyi, P., Klausberger, T. Defined types of cortical interneurone structure space and spike timing in the hippocampus. J Physiol. 562 (Pt 1), 9-26 (2005).
  6. Rudy, B., Fishell, G., Lee, S., Hjerling-Leffler, J. Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons. Dev Neurobiol. 71 (1), 45-61 (2011).
  7. Marin, O. Cellular and molecular mechanisms controlling the migration of neocortical interneurons. Eur J Neurosci. 38 (1), 2019-2029 (2013).
  8. Flames, N., et al. Short- and long-range attraction of cortical GABAergic interneurons by neuregulin-1. Neuron. 44 (2), 251-261 (2004).
  9. Zhu, Y., Li, H. -S., Zhou, L., Wu, J. Y., Rao, Y. Cellular and molecular guidance of GABAergic neuronal migration from an extracortical origin to the neocortex. Neuron. 23, 473-485 (1999).
  10. Zimmer, G., et al. Ephrin-A5 acts as a repulsive cue for migrating cortical interneurons. Eur J Neurosci. 28 (1), 62-73 (2008).
  11. Nobrega-Pereira, S., et al. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors. Neuron. 59 (5), 733-745 (2008).
  12. Lodato, S., et al. Excitatory projection neuron subtypes control the distribution of local inhibitory interneurons in the cerebral cortex. Neuron. 69 (4), 763-779 (2011).
  13. Elias, L. A., Turmaine, M., Parnavelas, J. G., Kriegstein, A. R. Connexin 43 mediates the tangential to radial migratory switch in ventrally derived cortical interneurons. J Neurosci. 30 (20), 7072-7077 (2010).
  14. Bellion, A., Baudoin, J. P., Alvarez, C., Bornens, M., Metin, C. Nucleokinesis in tangentially migrating neurons comprises two alternating phases: Forward migration of the Golgi/centrosome associated with centrosome splitting and myosin contraction at the rear. J Neurosci. 25 (24), 5691-5699 (2005).
  15. Marin, O., Valiente, M., Ge, X., Tsai, L. H. Guiding neuronal cell migrations. Cold Spring Harb Perspect Biol. 2 (2), a001834(2010).
  16. Martini, F. J., Valdeolmillos, M. Actomyosin contraction at the cell rear drives nuclear translocation in migrating cortical interneurons. J Neurosci. 30 (25), 8660-8670 (2010).
  17. Wamsley, B., Fishell, G. Genetic and activity-dependent mechanisms underlying interneuron diversity. Nat Rev Neurosci. , (2017).
  18. Chu, J., Anderson, S. A. Development of cortical interneurons. Neuropsychopharmacology. 40 (1), 16-23 (2015).
  19. Metin, C., Baudoin, J. P., Rakic, S., Parnavelas, J. G. Cell and molecular mechanisms involved in the migration of cortical interneurons. Eur J Neurosci. 23 (4), 894-900 (2006).
  20. Hernandez-Miranda, L. R., Parnavelas, J. G., Chiara, F. Molecules and mechanisms involved in the generation and migration of cortical interneurons. ASN Neuro. 2 (2), e00031(2010).
  21. Batista-Brito, R., et al. The cell-intrinsic requirement of Sox6 for cortical interneuron development. Neuron. 63 (4), 466-481 (2009).
  22. Marcorelles, P., et al. Evidence for tangential migration disturbances in human lissencephaly resulting from a defect in LIS1, DCX and ARX genes. Acta Neuropathol. 120 (4), 503-535 (2010).
  23. McManus, M. F., Nasrallah, I. M., Pancoast, M. M., Wynshaw-Boris, A., Golden, J. A. Lis1 is necessary for normal non-radial migration of inhibitory interneurons. Am J Pathol. 165 (3), 775-784 (2004).
  24. Pancoast, M., Dobyns, W., Golden, J. A. Interneuron deficits in patients with the Miller-Dieker syndrome. Acta Neuropathol. 109 (4), 400-404 (2005).
  25. Azzarelli, R., Oleari, R., Lettieri, A., Andre, V., Cariboni, A. In Vitro, Ex Vivo and In Vivo Techniques to Study Neuronal Migration in the Developing Cerebral Cortex. Brain Sci. 7 (5), (2017).
  26. Wang, Z. Z., et al. Chemokine-like factor 1 promotes the migration of rat primary cortical neurons by the induction of actin polymerization. Neuroreport. 25 (15), 1221-1226 (2014).
  27. Zito, A., et al. Neuritin 1 promotes neuronal migration. Brain Structure and Function. 219 (1), 105-118 (2014).
  28. Rakic, S., et al. Cdk5 phosphorylation of ErbB4 is required for tangential migration of cortical interneurons. Cereb Cortex. 25 (4), 991-1003 (2015).
  29. van den Berghe, V., et al. Directed migration of cortical interneurons depends on the cell-autonomous action of Sip1. Neuron. 77 (1), 70-82 (2013).
  30. Nery, F. C., et al. New methods for investigation of neuronal migration in embryonic brain explants. J Neurosci Methods. 239, 80-84 (2015).
  31. Vidaki, M., et al. Rac1-dependent cell cycle exit of MGE precursors and GABAergic interneuron migration to the cortex. Cereb Cortex. 22 (3), 680-692 (2012).
  32. Lysko, D. E., Putt, M., Golden, J. A. SDF1 reduces interneuron leading process branching through dual regulation of actin and microtubules. J Neurosci. 34 (14), 4941-4962 (2014).
  33. Tielens, S., et al. Elongator controls cortical interneuron migration by regulating actomyosin dynamics. Cell Res. 26 (10), 1131-1148 (2016).
  34. Shinohara, R., et al. A role for mDia, a Rho-regulated actin nucleator, in tangential migration of interneuron precursors. Nat Neurosci. 15 (3), S371-372 373-380 (2012).
  35. LoTurco, J., Manent, J. B., Sidiqi, F. New and improved tools for in utero electroporation studies of developing cerebral cortex. Cereb Cortex. 19, Suppl 1. i120-i125 (2009).
  36. De Marco Garcia, N. V., Fishell, G. Subtype-selective electroporation of cortical interneurons. J Vis Exp. (90), e51518(2014).
  37. Tobet, S. A., Hanna, I. K., Schwarting, G. A. Migration of neurons containing gonadotropin releasing hormone (GnRH) in slices from embryonic nasal compartment and forebrain. Dev Brain Res. 97 (2), 287-292 (1997).
  38. Stoppini, L., Buchs, P. -A., Muller, D. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods. 37, 173-182 (1991).
  39. Steinecke, A., Gampe, C., Valkova, C., Kaether, C., Bolz, J. Disrupted-in-Schizophrenia 1 (DISC1) is necessary for the correct migration of cortical interneurons. J Neurosci. 32 (2), 738-745 (2012).
  40. Friocourt, G., et al. Both doublecortin and doublecortin-like kinase play a role in cortical interneuron migration. J Neurosci. 27 (14), 3875-3883 (2007).
  41. Murthy, S., et al. Serotonin receptor 3A controls interneuron migration into the neocortex. Nat Commun. 5, 5524(2014).
  42. Nichols, A. J., O'Dell, R. S., Powrozek, T. A., Olson, E. C. Ex utero electroporation and whole hemisphere explants: a simple experimental method for studies of early cortical development. J Vis Exp. (74), (2013).
  43. Pacary, E., Guillemot, F. Cerebral cortex electroporation to study projection neuron migration. Curr Protoc Neurosci. 77, 2.26.21-22.26.18 (2016).
  44. Yozu, M., Tabata, H., Nakajima, K. The caudal migratory stream: A novel migratory stream of interneurons derived from the caudal ganglionic eminence in the developing mouse forebrain. J Neurosci. 25 (31), 7268-7277 (2005).
  45. Butt, S. J., et al. The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes. Neuron. 59 (5), 722-732 (2008).
  46. Miyoshi, G., et al. Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons. J Neurosci. 30 (5), 1582-1594 (2010).
  47. Zerucha, T., et al. A highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between Dlx genes in the embryonic forebrain. J Neurosci. 20 (2), (2000).
  48. Bottenstein, J. E. Cell culture in the neurosciences. , Plenum Press. (1985).
  49. Wang, Y., et al. Dlx5 and Dlx6 regulate the development of parvalbumin-expressing cortical interneurons. J Neurosci. 30 (15), 5334-5345 (2010).
  50. Zheng, H., et al. Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice. Anesthesiology. 118 (3), 516-526 (2013).
  51. Zhao, T., et al. Prenatal ketamine exposure causes abnormal development of prefrontal cortex in rat. Sci Rep. 6, 26865(2016).
  52. Timpe, J. M., Wang, C. Z., Kim, J., Johnson, K. M. Alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor activation protects against phencyclidine-induced caspase-3 activity by activating voltage-gated calcium channels. J Neurosci Res. 92 (12), 1785-1791 (2014).
  53. Myers, A. K., Meechan, D. W., Adney, D. R., Tucker, E. S. Cortical interneurons require Jnk1 to enter and navigate the developing cerebral cortex. J Neurosci. 34 (23), 7787-7801 (2014).
  54. Lopez-Bendito, G., et al. Chemokine signaling controls intracortical migration and final distribution of GABAergic interneurons. J Neurosci. 28 (7), 1613-1624 (2008).
  55. Close, J., et al. Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons. J Neurosci. 32 (49), 17690-17705 (2012).
  56. Anderson, S. A., et al. Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons. Neuron. 19 (1), 27-37 (1997).
  57. Stuhmer, T., Anderson, S. A., Ekker, M., Rubenstein, J. L. Ectopic expression of the Dlx genes induces glutamic acid decarboxylase and Dlx expression. Development. 129 (1), 245-252 (2002).
  58. Godin, J. D., et al. p27(Kip1) is a microtubule-associated protein that promotes microtubule polymerization during neuron migration. Dev Cell. 23 (4), 729-744 (2012).
  59. Rossokhin, A. V., Sharonova, I. N., Bukanova, J. V., Kolbaev, S. N., Skrebitsky, V. G. Block of GABA(A) receptor ion channel by penicillin: Electrophysiological and modeling insights toward the mechanism. Mol Cell Neurosci. 63, 72-82 (2014).
  60. Lindquist, C. E., Dalziel, J. E., Cromer, B. A., Birnir, B. Penicillin blocks human alpha 1 beta 1 and alpha 1 beta 1 gamma 2S GABAA channels that open spontaneously. Eur J Pharmacol. 496 (1-3), 23-32 (2004).
  61. Bortone, D., Polleux, F. KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner. Neuron. 62 (1), 53-71 (2009).
  62. Lin-Hendel, E. G., McManus, M. J., Wallace, D. C., Anderson, S. A., Golden, J. A. Differential mitochondrial requirements for radially and non-radially migrating cortical neurons: Implications for mitochondrial disorders. Cell Rep. 15 (2), 229-237 (2016).
  63. Lourenco, M. R., Garcez, P. P., Lent, R., Uziel, D. Temporal and spatial regulation of interneuron distribution in the developing cerebral cortex--an in vitro study. Neuroscience. 201, 357-365 (2012).
  64. Mahajani, S., et al. Lamin B1 levels modulate differentiation into neurons during embryonic corticogenesis. Sci Rep. 7 (1), 4897(2017).
  65. Liodis, P., et al. Lhx6 activity is required for the normal migration and specification of cortical interneuron subtypes. J Neurosci. 27 (12), 3078-3089 (2007).
  66. Close, J. L., et al. Single-cell profiling of an in vitro model of human interneuron development reveals temporal dynamics of cell type production and maturation. Neuron. 93 (5), e1035 1035-1048 (2017).
  67. Chen, Y. J., et al. Single-cell RNA sequencing identifies distinct mouse medial ganglionic eminence cell types. Sci Rep. 7, 45656(2017).
  68. Michaud, J. L., et al. The genetic landscape of infantile spasms. Hum Mol Genet. 23 (18), 4846-4858 (2014).
  69. Allen, A. S., et al. De novo mutations in epileptic encephalopathies. Nature. 501 (7466), 217-221 (2013).
  70. Bery, A., Merot, Y., Retaux, S. Genes expressed in mouse cortical progenitors are enriched in Pax, Lhx, and Sox transcription factor putative binding sites. Brain Res. 1633, 37-51 (2016).
  71. Nelson, B. R., Hodge, R. D., Bedogni, F., Hevner, R. F. Dynamic interactions between intermediate neurogenic progenitors and radial glia in embryonic mouse neocortex: potential role in Dll1-Notch signaling. J Neurosci. 33 (21), 9122-9139 (2013).
  72. Simon, R., et al. A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis. EMBO J. 31 (13), 2922-2936 (2012).
  73. Venkataramanappa, S., Simon, R., Britsch, S. Ex utero electroporation and organotypic slice culture of mouse hippocampal tissue. J Vis Exp. (97), (2015).
  74. Machacek, M., et al. Coordination of Rho GTPase activities during cell protrusion. Nature. 461 (7260), 99-103 (2009).

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