1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies
Hurtado de Mendoza, T., Balana, B., Slesinger, P. A., Verma, I. M. Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection. J. Vis. Exp. (51), e2564, doi:10.3791/2564 (2011).
Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.
1. Organotypic Cerebellar Cultures:
2. Fas Treatment and Staining of Cerebellar Slices:
3. Representative Results:
The main challenge of this protocol is to be able to generate healthy cerebellar slice cultures, especially since our final readout will be cell death. A healthy culture appears as one where the cell body layer is translucent and allows you to see the foliated structure of the cerebellum under the microscope (Figure 1). After few days in culture the slices start to thin and non-neuronal cells can be observed migrating away from the margins of the slice. The dark round cells (most likely macrophages) that cover the slices after few days in culture, will eventually decrease in number after 10-14 days in vitro (Figure 2) 5. The ultimate proof for the viability of the slices is to stain for different cellular markers, such as Calbindin for the Purkinje cells. Figure 3 shows a representative confocal microscope image of the Purkinje cell layer with several TUNEL positive nuclei.
It is important to consider that even if some slices received an apoptotic stimulus, they were only exposed for 24 hours. This allowed sufficient time to observe DNA fragmentation in the dying cells with the TUNEL staining, but a defined Purkinje cell layer was still present.
Figure 1. Healthy cerebellar slice at DIV2. This image shows the translucent cell body layer and the foliated cerebellar structure in a healthy slice.
Figure 2. Healthy cerebellar slice at DIV 7. In this image we can still observe the foliated structure of the cerebellum and the appearance of dark cell bodies.
Figure 3. Representative image of a Fas-treated cerebellar slice. This confocal image shows the Purkinje cells stained with Calbindin (green) and the apoptotic cells positive for TUNEL staining (red). Purkinje cells do not appear in a single row but are clustered forming a folium-like structure.
This method describes one of the many possible applications of neuronal organotypic cultures and it has allowed us to study the effect of apoptotic stimuli in wild type and mutant cerebellar slices.
The most critical part of this experiment is to be able to consistently generate healthy cerebellar slice cultures. The key factors are dissection and slicing technique and the ability to perform the protocol in the least time possible, but at the same time being careful not to damage the slices. Another crucial factor is the preparation and composition of the culture media, these cultures are very sensitive, so we had to test several recipes and brands of media. Even different batches of serum or any other components of the media could affect the viability of the slices. We got the best results with the Horse Serum from Invitrogen Lot # 480116.
Once the cerebellar slices are generated one can study cerebellar development, electrophysiology, cell survival alone or in response to apoptotic stimuli, excitotoxicity or oxygen deprivation. Comparative studies using wild type and mutant cerebellar slices have been very insightful in many aspects of cerebellar function and development.
All animal work conducted was done in accordance with policies established by the IACUC. The Salk Institute is an AAALAC accredited facility.
This study was funded in part by the IPSEN Foundation. IMV is an American Cancer Society Professor of Molecular Biology, and holds the Irwin and Joan Jacobs Chair in Exemplary Life Sciences. This work was supported in part by grants from the NIH, Leducq Foundation, Meriaux Foundation, Ellison Medical Foundation, Ipsen/Biomeasure, Sanofi Aventis, Prostate Cancer Foundation, and the H.N. and Frances C. Berger Foundation. PAS is funded by McKnight Endowment Fund for Neuroscience and the National Institute on Drug Abuse (R01 DA019022). The authors would like to thank Mark Schmitt. Graphic art was designed by Jamie T. Simon .Sponsorship was kindly provided by Invitrogen.
|6 well / 24 well cell culture plates||Corning||3506/ 3527|
|30mm culture plate inserts (0.4-Ám pore)||EMD Millipore||PICM03050|
|Purified NA/LE Hamster anti-mouse CD95 (Jo2)||BD Biosciences||554254|
|In situ cell death detection kit, TMR red||Roche Group||12 156 792 910|
|Rabbit anti-Calbindin D-28k antibody||Swant||CB-38a|
|Alexa 488-goat anti-rabbit IgG secondary antibody||Invitrogen||A-11008|
|Vectashield Mounting Medium with DAPI||Vector Laboratories||H-1200|
|Dissecting Microscope||Carl Zeiss, Inc.|
|Confocal Microscope||Leica Microsystems|