Further Shenanigans in the Murine Cerebellum

By Vivian Wan

My work over the past weeks has been focused on studying the possible influence of the duplicated chromosomal region (in our autistic mouse model) on cerebellar Purkinje Cell degeneration or development issues.

Golgi stained murine cerebellum sections.

After a long wait for the results of our Golgi staining trials, we finally were able to see the fruits of our efforts under the microscope. As shown in the picture, we sectioned the Golgi stained cerebelli into thick slices and mounted them on glass slides. To analyze images of Purkinje Cells from these sections, I use a program called ImageJ. Specifically, I use ImageJ plugins NeuronJ and Scholl Analysis for tracing of dendrites and for branching analysis [1], respectively. The former, NeuronJ, detects the path of a dendrite as you trace it, making it easier for tracing as opposed to tracing freehand. From these tracings, I have been gathering data on the sum of the dendritic arbor lengths.

I am also quantifying branching of the dendritic arbor. One way to do this is with the second plugin, Sholl Analysis, which quantifies intersections of dendrites within a certain area [2]. The program analyzes the number of intersections within concentric circles of increasing diameter from a chosen point — in our case the cell body — and outputs a plot, showing the number of branches enclosed within the ring-like area as a function of the distance from the chosen point. The second way to measure branching requires more particular definitions of dendrite branches. For Purkinje Cells, there are proximal dendrites, which, in addition to other functional characteristics, have sparse numbers of spines, and distal dendrites, which exhibit a high density of spines. For this second method, I categorized branching by orders: the first order were distal dendrites branching from the proximal dendrites.

10x microscopic view of a Golgi stained Purkinje Cell.

I also analyzed the Purkinje Cells in other criteria, such as measuring the distance from the start of the dendritic arbor to the end and measuring the sum of the length of the proximal dendrite. We are also considering looking at spine density (from highly magnified images) as a measurement.

On a different note, we also obtained more promising data on protein expression levels from our autistic mouse model. As exciting as it is to find a direction in which to pursue more research, as it often goes, new results only lead to more questions. Challenges may be, for instance, apparent contradictions with previous research, more controls may be necessary in light of the new results, or the new results may even challenge the original hypothesis, demanding new interpretations. How can the problems be resolved? In our case, we will continue to explore our mouse model by looking at our data with more methods. Unfortunately, as I have come to the end of my fellowship, the questions will be left open on my end. But I am thrilled to have contributed to this project and await further findings by others.

References:

[1] Grasselli et al., PLoS One. 2011;6(6):e20791.

[2] Sholl DA, J Anat. 1953 Oct;87(4):387-406.