Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The overall goal of this procedure is to target genetic modification to the external germinal layer of the chick cerebellum to observe, examine and modify granule cell morphology and behavior in an ex vivo setting. This method can help answer key questions in the developmental neurobiology field, such as how neural progenitors migrate, proliferate, and differentiate. The main advantage of this technique is that it carries with it the considerable benefits in cost and convenience that are associated with studies.

Though this method can provide insight into chicken granule development, it can also be applied to other model organisms such as reptiles and mammals. To begin place fertilized brown chicken eggs into an egg incubator at 38 degrees Celsius until they reach embryonic. Day 14 on embryonic day 14, cut an opening in the egg to expose the embryo and decapitate the chick embryo in ovo.

Placed the head into a Petri dish containing ice cold PBS under dissecting microscope. Use standard forceps to make an incision behind each eye all the way through the tissue and removing the eyes and upper jaw. Then make a second incision all the way through the pharynx to remove the lower jaw.

Next, use standard forceps to remove the skin from the surface of the skull by peeling it away. Then remove the frontal and parietal bones revealing the brain from a ventral aspect. Remove the pharyngeal cartilage and the auxiliary mesenchyme.

Then place the brain dorsal side up and carefully remove the mesenchyme dorsal to the hind brain, taking care not to damage the p. Next, make an incision all the way through the tissue between the midbrain and the hind brain to detach the hind brain, including the cerebellum. Make incisions all the way through the tissue at both the lateral junctions of the cerebellum and the aller plate of the hind brain.

Remove the entire cerebellum taking care to maintain the integrity of the PIA throughout the dissection. Finally, remove the forming choroid plexus and move the dissected cerebellum into ice cold HBSS. Transfer the whole cerebellum to the sterile platform of a tissue chopper using a spatula or a three milliliter pasture pipette with a tip cut away.

To widen the aperture, remove any excess liquid using a pipette. Set the cutting speed of the tissue chopper to 50%of its maximum value. Then cut the cerebellum in the required orientation at a thickness of 300 micrometers using a three milliliter pasture pipette.

Cover the sliced cerebellum in cold HBSS. Then transfer the HBSS and the slices to a 60 millimeter Petri dish containing 20 milliliters of ice cold HBSS using the three liter pasture pipette with a cut tip, place a Petri dish under a dissecting microscope illuminated with a fiber optic light source. Then use a watchmaker forceps to separate individual slices of brain tissue and identify the slices to be electroporated based on their tissue integrity and media lateral position.

Construct an electroporation chamber by fixing the anode of an electro to the base of a 60 millimeter Petri dish. Using insulation tape, add approximately one milliliter of HBSS to cover the electrode. Next place, a 0.4 micrometer culture.

Insert on top of the electrode covered in HBSS. Use a three milliliter pasture pipette with a cut tip to transfer the identified slices up to five per insert onto the culture insert. Then separate the slices and allow them to settle onto the culture.

Insert in a sagittal orientation. Using a pipette, remove excess medium from the brain slices. Allow the insert to rest on the electrode so that there is contact between the insert and the electrode.

In this setup. The culture insert with the slices will rest upon the surface of the medium, maintaining the circuit, but allowing spatial targeting of the cathode. Using a P 10 pipette tip pipette five microliters of DNA diluted with 20%fast green over the surface of a targeted region of each slice.

The addition of fast green ensures that the DNA solution is viscous enough to prohibit wide dispersal of the DNA. Then place the cathode of the desired target tissue and electrolyte the samples. Avoid direct contact of the cathode with the tissue by placing the cathode as close to the tissue as possible without actually touching it.

Repeat the electroporation in multiple regions of the external granule layer on each individual cerebellar slice as desired. When finished, transfer the culture insert to a 30 millimeter Petri dish To each culture. Add one milliliter of prewarm culture medium underneath the culture insert.

Make sure that the insert does not float on the medium. The culture insert should be in contact with the medium, but the slices should not be bathed in it. Incubate the cultures for up to three days replacing all of the culture.

Medium every 24 hours with fresh prewarm, medium following culture. Transfer the slices on the culture inserts to a 30 millimeter dish containing 4%para formaldehyde for one hour. Make sure to add the para formaldehyde on top of the culture.

Insert once fixed. Wash the slices three times in PBS for five minutes each. Then use ophthalmic scissors to dissect each of the electroporated and cultured cerebellar slices from the culture.

Insert by cutting around each slice and removing it along with the attached region of the insert. Do not attempt to remove the slice from the insert surface. Mount the slices in approximately one milliliter of a mounting medium of choice under a cover slip while taking care not to introduce bubbles.

Once the slides are dry, collect the slides and image them using laser scanning. Confocal microscopy calbindin. Staining of E 14 cerebellar tissue is shown here in red following electroporation with a control GFP plasmid at one, two, and three days in vitro.

The consistent calbindin staining shows that tissue integrity is maintained in culture for at least three days in vitro. Here are low magnification images of RFP and GFP encoded plasmids that were successfully electroporated into the external granule layer at various locations. The GFP plasmid was targeted to a single fum as indicated by the asterisk.

An example is shown here where a construct encoding GFP driven by an 8 0 1 enhancer has been electroporated into the external granule layer. The expression of 8 0 1 defines granule cell precursors within the external granule layer. Various cell morphologies are clearly visible at three days in vitro and cell behavior can be monitored.

After watching this video, you should have a good understanding of how to elect prate individual cerebellar slices with a variety of genetic tools to observe and manipulate granule cell development. Following this procedure, other methods like immunohistochemistry can be performed in order to answer additional questions about the structural integrity of the tissue and relative locations of different cell types such as Perkin cells or Bergman gl Once mastered. This technique can be done in about two hours for a dozen chicken embryos if it's performed properly.