Anatomical Institute, Department of Biomedicine, University of Basel
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Kapfhammer, J. P., Gugger, O. S. The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures. J. Vis. Exp. (61), e3637, doi:10.3791/3637 (2012).
Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.
1. Setting up Organotypic Cerebellar Slice Cultures
Cerebellar slice cultures are prepared from postnatal day 8 P(8) mouse pups using the static incubation method 10. In our laboratory, we use B6CF1 mice. In some experiments also transgenic mice were used which express EGFP selectively in Purkinje cells. The preparation of slice cultures takes approximately 30 minutes per mouse pup, i.e. 3 hours for a litter of 6 mouse pups. All steps are carried out under sterile conditions in a laminar flow workbench with sterilized surgical instruments.
2. Fixation and Immunostaining of Organotypic Cerebellar Slice Cultures
An important advantage of the cerebellum is that antibodies are available that specifically and brightly stain the principal cerebellar neurons, i.e. Purkinje cells and granule cells. The dendritic morphology of Purkinje cells can be revealed by immunostaining with anti-calbindin D28K.
3. Viewing of Individual Purkinje Cells in Organotypic Slice Cultures
4. Measurement of Purkinje Cell Dendritic Tree Size and Branch Points
5. Representative Results
Monitoring the development of the Purkinje cell dendritic tree during the culture period
Organotypic slice cultures derived from Pcp2-EGFP mice which express EGFP specifically in Purkinje cells allow to study the morphology of individual cells during several days in culture. This way the growth and development of the dendritic tree during the culture period can nicely be documented. Living identified Purkinje cells were photographed every 2nd or 3rd day during the culture period with the 10x objective. This low power objective was chosen to avoid and minimize phototoxic damage to the cells due to the illumination with fluorescent light. Figure 3 shows two examples of Purkinje cells photographed in living cultures. The first cell was followed from 2 days in vitro until 7 days in vitro (Figure 3, A-C). It is evident that the dendritic tree of this cell during this time period grows several new branches and expands in size. In the second example, a Purkinje cell was followed from 4 to 11 days in vitro (Figure 3, D-F). The small dendritic tree present at DIV4 expands substantially during this time period. Both examples show that most of the dendritic tree of Purkinje cells present at the end of the culture period did indeed develop in the culture.
Development of the Purkinje cell dendritic tree is inhibited by PKC or mGluR1 stimulation
Organotypic cerebellar slice cultures were cultured for 11 days. Starting at the 2nd day in vitro, some of the cultures were treated with either PMA (50 nM), a phorbol ester stimulating PKC or DHPG (10 μM), a mGluR1 agonist, until the cultures were fixed. Both compounds have been shown previously to inhibit and limit Purkinje cell dendritic growth 7, 8, 9, 5. In the untreated control slices Purkinje cells developed a typical large and highly branched dendritic tree which was visualized either by anti-Calbindin immunostaining (Figure 4A) or in cultures with EGFP-expressing Purkinje cells, by immunostaining with anti-GFP (Figure 4D). In cultures treated with PMA the morphology of the dendritic tree was profoundly altered. The dendrites appeared thickened and had only few short side branches. Many Purkinje cells, unlike in control cultures, were no longer unipolar, with one primary dendrite emanating from the cell body, but developed two or even more primary dendrites (Figure 4B, E). The territory covered by the dendritic tree was markedly reduced (see below). A similar situation was present in DHPG-treated cultures. The dendritic tree of the Purkinje cells was greatly reduced in size and the branching was markedly reduced (Figure 4C, F). However, there were also some qualitative differences compared to PMA-treated cultures. There was no thickening of the dendritic branches, and the primary dendrites carried many very short secondary dendrites (Figure 4C, F) suggesting that the signaling events going on in PMA and DHPG-treated Purkinje cells may be similar, but not identical.
Quantitative evaluation of Purkinje cell dendritic tree size and branch points For quantitative evaluation, the size of the Purkinje cell dendritic tree and the number of branch points per Purkinje cell is measured as described above. Results from at least three independent experiments are pooled together, and a minimum of 20 cells need to be analyzed. The mean of the area covered by the dendritic trees for the control experiments is determined and set as 100%, and the values for the other experiments are expressed as percent values accordingly. Statistical analysis of the data is done with GraphPad Prism software. Because we do not assume that all values determined are part of a Gaussian distribution, we use statistical tests that do not assume such a distribution of the measured values. For comparing multiple conditions and testing for statistical significance, we use Kruskal-Wallis test followed by an appropriate procedure for post hoc tests for rank-based statistics, as, e.g., implemented in the Dunn's post test in GraphPad Prism. Results are typically presented as bar graphs. An example of such a statistical evaluation is given in Figure 5. Both the analysis of the dendritic tree size (Figure 5A) and the number of branch points per Purkinje cell (Figure 5B) show clear differences between the control condition and DHPG or PMA treatment. Both parameters were different with a statistical significance of p<0.001 according to the Kruskal-Wallis test.
Figure 1. EGFP-labeled Purkinje cell. In Pcp2-EGFP mice not all Purkinje cells express EGFP. Therefore, dendritic trees of some Purkinje cells can be viewed individually when they express EGFP (EGFP channel in A) and the neighboring Purkinje cells with overlapping dendritic trees do not (negative in EGFP channel shown in A, positive in the anti-Calbindin staining shown in the red channel in B). Scale bar = 50 μm.
Figure 2. Measurement of dendritic parameters of Purkinje cells. (A). The size of the dendritic tree can easily be measured by tracing the outline of the Purkinje cell with a single mouse click in the Image analysis software Image Pro plus using the magic wand tool. The magic wand tool is used such that the entire dendritic tree including the cell soma and all dendritic branches are completely encircled by the line. (B). The number of branch points is counted manually in images of Purkinje cells. Every branch point is marked with a yellow dot. Scale bar = 50 μm.
Figure 3. Monitoring the development of the Purkinje cell dendritic tree. Identified Purkinje cells were photographed repeatedly to monitor the growth of the dendritic tree. (A-C): Dendritic tree of a Purkinje cell growing and developing from day in vitro (DIV) 2 until DIV7. There is continuous growth and branching of the dendrites during this culture period. (D-F): Dendritic tree of a Purkinje cell growing and developing from DIV4 until DIV11. The dendritic tree expands substantially during this culture period. Scale bar = 50 μm.
Figure 4. Development of the Purkinje cell dendritic tree is inhibited by PKC or mGluR1 stimulation. (A), (D): Untreated control cells with a well developed dendritic tree. (B), (E): After 9 days of PMA treatment dendrites appear thickened and the dendritic tree is strongly reduced in sized. Cells often lose their polarization and become bipolar (E). (C), (F): After 9 days of DHPG treatment the dendritic tree is greatly reduced in size. Unlike in the PMA treatment, many very fine and short side branches are present on the primary and secondary dendrites. Cells often lose their polarization and become bipolar (F). Purkinje cells were visualized by anti-Calbindin immunostaining in (A - C) and by EGFP-Expression in cultures derived from Pcp2-EGFP mice in (D - F). Scale bar = 50 μm.
Figure 5. Quantitative evaluation of Purkinje cell dendritic tree size and branching. Quantitative measurements show that both dendritic tree size (upper bar graph) and the number of branch points (lower bar graph) are greatly reduced after PMA or DHPG treatment. Differences between control dendritic trees and dendritic trees of Purkinje cells from treated cultures were significant with p<0.001.
The methods presented here allow to study Purkinje cell dendritic development in organotypic cerebellar slice cultures and to quantitatively evaluate Purkinje cell dendritic expansion by measuring dendritic tree size and the number of dendritic branch points. Of course, a more extensive and sophisticated quantitative analysis of Purkinje cell dendrites is possible, e.g. by determining total dendritic length, performing a Sholl analysis or determining the fractal dimension of the dendritic tree. For this type of analysis it is usually required to manually trace the entire Purkinje cell dendritic tree into an analysis program as for example Neuro-Lucida. While these more refined methods certainly yield a superior level of analysis (e.g. see 1, 6), they are more time consuming and require more specialized equipment and analysis software compared to the methods presented here. For most applications, in particular in situations where the changes in the Purkinje cell dendrites are considerable and qualitatively visible, the simple methods presented were will be sufficient. It should be noted that the organotypic cerebellar slice cultures derived from P8 mice will cover only the later phase of Purkinje cell dendritic development characterized by rapid dendritic expansion. For the study of earlier stages, the slice culture method needs to be adapted to slices from neonatal or embryonic animals which then will cover the earlier phases of dendritic development (e.g. 2).
For the visualization of Purkinje cells in organotypic cerebellar slice cultures, we have focused only on two routine methods: the anti-Calbindin immunostaining and the use of mice with EGFP expressing Purkinje cells. Of course more methods are available, in particular biolistic or viral transfection with fluorescent proteins, diolistic labeling with fluorescent dyes and intracellular dye injections into single Purkinje cells. Depending on the requirements of the study and the equipment available in the laboratory these methods can significantly enhance the level of analysis of Purkinje cell dendritic morphology, for example by analyzing dendritic spines using confocal or two photon microscopy.
Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and permitted by Swiss authorities. The authors have nothing to disclose.
This work was supported by the University of Basel, Department of Biomedicine, and the Swiss National Science Foundation (31003A-116624).