Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful tool for studying neurodevelopmental and degenerative or regenerative processes. This technique can be used to rapidly screen candidate molecules for their neuroprotective potential. This method closely mimics in vivo conditions, compared to dissociated primary cell cultures, as the tissue set architecture and native cell-cell connections are preserved within the planes of the sections.

Here we demonstrate the study of Purkinje cell death in the developing cerebellum. But organotypic slice culture is equally suitable to model neurodegenerative diseases in almost every central nervous system region. Demonstrating the procedure with Jennifer Rakotomamonjy will be Sean McDermott, a technician from my laboratory.

Before harvesting the cerebellar slices, in a sterilized biosafety cabinet, fill each well of a six-well culture plate with one milliliter of vacuum-filtered culture medium, and add the pharmacological agent of interest to the treatment wells, and an equal volume of vehicle to the control wells. Using sterile forceps, place one 0.4-micrometer pore size PTFE membrane filter insert into each well, taking care to avoid bubbles at the interface of each membrane and the medium, and equilibrate the medium in a 37 degree Celsius and 5%carbon dioxide incubator for two hours. To harvest the cerebellum, use straight dressing forceps to grasp the pup head by the nose, and use straight eye scissors to cut open the scalp from the posterior end laterally to the midline.

Cut open the skull in the same manner, pointing the scissor tips outward to avoid damaging the cerebellum, and use a spatula to transfer the brain to a 60-millimeter dish containing cold HBSS and five milligrams per milliliter of glucose. Use the straight dressing forceps to carefully dissect out the cerebellum, and use sterile curved fine forceps to place the tissue onto a plastic disc on the cutting table of a tissue chopper. Rotate the cutting table to orient the tissue to allow the acquisition of parasagittal sections, and pull the table release knob to the right to move the cutting table until the blade is positioned at the edge of the tissue.

Then adjust the slice thickness to 350 micrometers, and the blade speed to medium, and start the chopper. When the whole cerebellum has been sliced, turn off the chopper. Using sterile forceps, hold the disc over a new 60-millimeter dish.

Use a transfer pipette to flush HBSS plus glucose over the disc so that the slices fall into the dish. Then, touching the samples as minimally as possible, use a microprobe to separate the slices. Use the transfer pipette to select sections of the cerebellum close to the vermis onto individual cell culture inserts in the six-well plate, and use the microprobe to position the slices into the center of each insert.

When all of the slices have been placed, carefully aspirate any excess HBSS plus glucose, and return the plate to the cell culture incubator. For immunofluorescence staining, remove the supernatant from each well, and wash the inserts with PBS. Fix the slices with one milliliter of cold 4%paraformaldehyde in the well of each insert, and 500 microliters on top of each insert for one hour.

At the end of the fixation, wash the inserts four times for 10 minutes with one milliliter of fresh PBS under each insert and 500 microliters of fresh PBS on top of each insert per wash on an orbital shaker. Pre-fill each well of a 24-well plate with 500 microliters of PBS-TB, and use a paintbrush to transfer the cerebellar slices from each cell culture insert into individual wells of a 24-well plate. Permeabilize and block the slices at room temperature for one hour.

At the end of the incubation, label the cells with 200 microliters of the primary antibody of interest diluted in PBS-TB per well overnight at four degrees Celsius on the orbital shaker. The next morning, wash the slices four times for 10 minutes and 500 microliters of fresh PBS per wash on the shaker, before labeling the samples with the appropriate fluorophore-conjugated secondary antibodies for two hours at room temperature on the shaker, protected from light. At the end of the incubation, counterstain the sections with 500 microliters of an appropriate nuclear stain per well for 10 minutes at room temperature, protected from light.

And use a transfer pipette to mount the slices onto glass slides. Then let the sections air dry completely before rehydrating with PBS and mounting the tissues with coverslips coated with approximately 80 microliters of mounting medium. Once the mounting medium has cured, the cerebellar sections are ready to be imaged.

At postnatal day six, Purkinje cell survival is low in the control samples, consistent with their known vulnerability window. Survival increases as the donor animal grows older and exits this critical period. The treatment of cerebellar slices with a high concentration of potassium chloride results in successful induction of their depolarization and survival.

To obtain consistent and reproducible results, it is critical to select healthy cerebellar sections, and to perform the culture setup as efficiently as possible. Post-culture applications go beyond immunofluorescence. As organotypic slices can be used for genome protein expression studies, and for monitoring neuronal circuit activity using electrophysiology and calcium live imaging.