Neuron, 59(3), 425C438. characteristics, such as the soma area and the dendritic field area, and Sholl analysis of ON cells and OFF cells revealed significant differences between each region. We classified SMI\32+ RGCs into five clusters based on morphological features and found that a majority of SMI\32+ RGCs belong to alpha\like cells; however, a small proportion of SMI\32+ RGCs had small soma and small dendritic fields. Together, we present a full description of the morphology and distribution of SMI\32 immunoreactive RGCs in the rat retina. Image J plug\in was used to manually trace the dendritic arbor of each cell, and the rendered paths images were generated as previously described (Iaboni et?al., 2020). Parameters were measured as follows(1) soma area: area of cell body; (2) diameter of soma: the longest diameter of cell body; (3) dendritic field area: the area made from joining the ends of each terminal dendritic branch together using straight lines; (4) arbor asymmetry: the ICI 211965 distance between the center of mass of dendritic density and the cell body position; (5) total branches: total number of branches; (6) total dendritic length: the sum of the length of all branches. The soma area, diameter of soma, and dendritic field were calculated using the ImageJ measure function. Asymmetry, number of branches, and total dendritic length were calculated based on the rendered paths image. Sholl analysis was performed on the rendered paths image generated above with the Sholl ImageJ plug\in as described before (Ferreira et?al., 2014). 2.7. Cluster analysis To classify the SMI\32+ RGCs, we performed unsupervised clustering based on the six measured parameters, including ON OFF property, soma area, dendritic field area, arbor asymmetry, total branches, and total dendritic length. Clustering was implemented with custom MATLAB (The MathWorks Inc, Natick, MA) scripts and the built\in hierarchical clustering function (MATLAB Statistics Toolbox). Before clustering, each parameter was normalized by the is the is the mean value of is the standard deviation of represents the .05; ** .01) TABLE 2 Morphological parameters of all 83 neurobiotin\filled SMI\32+ RGCs thead th align=”left” rowspan=”1″ colspan=”1″ /th th align=”left” rowspan=”1″ colspan=”1″ /th th align=”center” rowspan=”1″ colspan=”1″ /th th style=”border-bottom:solid 1px #000000″ colspan=”3″ align=”left” rowspan=”1″ ON, OFF /th th align=”left” rowspan=”1″ colspan=”1″ /th th align=”left” rowspan=”1″ colspan=”1″ /th th align=”left” rowspan=”1″ colspan=”1″ /th th align=”left” rowspan=”1″ colspan=”1″ /th th align=”left” rowspan=”1″ colspan=”1″ Diameter of soma (m) /th th align=”left” rowspan=”1″ colspan=”1″ em n /em /th th align=”left” rowspan=”1″ colspan=”1″ ON /th th align=”left” rowspan=”1″ colspan=”1″ OFF /th th align=”left” rowspan=”1″ colspan=”1″ ON\OFF /th th align=”left” rowspan=”1″ colspan=”1″ Soma area (mean SEM, m2) /th th align=”left” rowspan=”1″ colspan=”1″ Dendritic field area (mean SEM, 103 m2) /th th align=”left” rowspan=”1″ colspan=”1″ Total dendritic length (mean SEM, mm) /th /thead Central 20413C192.41 19.5081.57 6.544.56 0.4920C3013652353.35 28.43155.40 17.856.54 0.7230954C530.02 25.18151.39 26.996.18 0.45Middle 20431C157.92 25.5881.20 15.634.92 0.2120C3014311C338.08 20.28102.62 7.625.39 0.30301055C521.51 21.18146.71 12.726.82 0.35Peripheral 20321C239.91 12.3898.60 6.795.94 0.7220C3016952385.24 20.59153.79 17.115.87 0.28?301037C543.19 37.27173.19 17.927.71 0.37 Open in a separate window To further investigate different characteristics in different parts of the retina, we compared SMI\32+ RGCs between the central, middle, and peripheral regions and also between ON cells and OFF cells (ONCOFF cells were not included). We found that in the central retina, both the soma area (Figure?2e) and the dendritic field area (Figure?2f) of ON cells were larger than that of OFF cells, but not in COL4A6 the middle or peripheral retinal regions. The dendritic field area of OFF cells at the periphery was larger than at the center, a pattern not observed in ON cells (Figure?2f). There was no significant difference between total dendritic length between different retinal regions or between ON and OFF cells (Figure?2g). 3.3. Arbor asymmetry of SMI\32+ RGCs Arbor asymmetry refers to the distance between the center of ICI 211965 mass of dendritic density and the position of the cell body (Bae et?al., 2018; Ran et?al., 2020). We measured all 83 neurobiotin\filled SMI\32+ RGCs and found that arbor asymmetry ranged approximately from 0 to 100. Arbor asymmetry was not significantly correlated with the soma area ( em r2? /em =?.0009401; Figure?3a), and there was no significant difference in arbor asymmetry between different retinal regions, nor between ON and OFF cells (ON\OFF cells ICI 211965 not included; Figure?3b). Furthermore, both ON cells and OFF cells were distributed in each arbor asymmetry interval (Figure?3c,d). These results suggest that subsets of SMI\32+ RGCs may have both symmetric and asymmetric arbor morphologies. Open in a separate window FIGURE 3 Arbor asymmetry of SMI\32+ RGCs. (a) Linear regression analysis between soma area and arbor asymmetry ( em r2? /em =?.0009401)..