Abstract
Purpose :
The dendritic arbor of retinal bipolar cells is vital for appropriate signal transduction, but quantitative data on the arbor complexity from young and aged mice remains unexplored. Based on the fractal repetition of the dendrites at different size scales, we introduced fractal dimension, D, as a mathematical measure to quantify the spatial complexity of dendrites and their ability to connect to photoreceptors. This study tests the hypothesis that two-month old healthy mice will have retinal bipolar cell dendritic arbors with an average D of approximately 1.40.
Methods :
C57BL/6J mice were obtained from the Jackson laboratory. After euthanizing, eyes were fixed with 4%PFA/PBS and 16μm cryosections were prepared. After immunostaining the bipolar cells with anti-PKCα antibody from Sigma Aldrich and goat anti-rabbit IgG secondary antibody, the images were obtained with Zeiss LSM 880 spinning disk confocal microscopy. 50 scans from 4 different 8-week-old C57BL/6J mice (2 males and 2 females) were obtained, for a total scan number of N=200. Fractal dimension, D, was calculated by rendering a 3D model of the arbor, then MATLAB was used to voxelize and perform a box-count of the dendrites. D values were optimized using the connectivity-cost optimization curve.
Results :
We successfully stained the mouse retinal bipolar cells, in which the high quality of fluorescent imaging enables us to calculate the D value. Cells were scanned via video and 2D still images, overlapping cells that could not be distinguished were excluded from data. Scans showing the entire arbor with limited background noise were included in data. D values were found with a minimum of 1.30, a maximum of 1.47, an average of 1.45, and a standard deviation of ±0.2. This value is similar to our previous investigation of pyramidal neurons in the coronal plane of the dorsal hippocampus.
Conclusions :
The fractal dimension D of retinal bipolar cells in young C57BL/6J mice was successfully measured. Notably, the value closely aligned with those of other healthy neurons we have examined. This fact can also imply that D values of neurons can be useful for a quantitative measure of arbor complexity and understanding the status of the neuron. Currently, we are obtaining D values from retinal bipolar cells of the aged (6 months) C57BL6J mice to examine how age dependency affects arbor fractality.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.