In situ quantification of nucleus and soma size
Coronal brain slices from Nr5a1-Cre;Ai14, Scnn1a-Tg3-Cre;Ai14, and Rbp4-Cre_KL100;Ai14 mice were stained with anti-dsRed (Clontech #632496) to label tdTomato and DAPI to label nuclei. Maximum intensity projection from six confocal stacks of 1-µm intervals were processed for analysis. Initial segmentation was performed using CellProfiler \cite{Lamprecht2007} to identify nuclei using DAPI signal and soma using tdTomato signal. Segmentation results were manually verified and any mis-segmented nuclei or somas were removed or re-segmented if appropriate. Area measurement of segmented nuclei and somas was performed in CellProfiler in Layer 4 from Nr5a1-Cre;Ai14 and Scnn1a-Tg3-Cre;Ai14 mice and Layer 5 from Rbp4-Cre_KL100;Ai14 mice.
For measurements of nucleus and soma size agnostic to Cre driver, 16 µm tissue sections from P56 mouse brain were used. To label nuclei, DAPI was applied to the tissue sections at a final concentration of 1mg/ml. To label the soma of cells, tissue sections were stained with Neurotrace 500/525 fluorescent Nissl stain (ThermoFisher Scientific) at a dilution of 1:100 in 1X PBS for 5 minutes, followed by brief washing in 1X PBS. Sections were coverslipped with Fluoromount-G (Southern Biotech) and visualized on a Nikon TiE epifluorescence microscope using a 40x oil objective. Soma and nuclei area measurements were taken by tracing the boundaries of the Nissl-stained soma or DAPI-stained nucleus, respectively, using cell measurement tools available in the Nikon TiE microscope software. All cells with a complete nucleus clearly present within the section were measured, except that we excluded glial cells which had very small nuclei and scant cytoplasm. Measurements were taken within a 40x field of view across an entire cortical column encompassing layers 1-6, and the laminar position of each cell (measured as depth from the pial surface) was tracked along with the nucleus and soma area measurements for each cell.
A linear regression was fit to nuclear versus soma area, and the slope was raised to the 3/2 power to estimate the nuclear proportion. This transformation was required to convert area to volume measurements and assumed geometric similarity of soma and nuclei.
Code availability
Acknowledgements
Competing interests
The authors declare no competing interests.
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