Measurement of LGN Soma Size

For each animal, the coronal sections of the LGN at the middle of the anterior-posterior axis, which correspond to the horizontal meridian of the visual field (Sanderson, 1971), were selected for soma size measurement. Regions-of-interest (ROIs) of 243 μm × 183 μm were set at six sites in the medial half of layers A and A1 of the LGN. Images of each ROI were captured using a microscope (BZ-9000, Keyence, Japan) with a × 60 microscope objective through the full thickness of each section at a 2 μm step. Cells with dark cytoplasmic and nucleolar staining and pale nuclear staining were considered as neurons (Duffy et al., 2014). They were all sampled for area measurement in each ROI (228–457 cells in each layer). Neuron size was measured by delineating soma using the image processing software Fiji (Schindelin et al., 2012) at the depth in which cell nucleoli were clearly observed, thus providing the cell's maximum diameter at the presumed soma midline. Soma size measurement was performed in blind condition. The images of the specimen were given to the measurers without the information about the rearing condition or LGN layer.

The areas of all measured soma in individual ROIs were averaged and the average of all ROIs in each layer of individual animals was used as the representative value of the layer. The difference in soma size in layers A and A1 of individual animals was evaluated by calculating the shrinkage index as follows:

where A and A1 are the representative values of soma size in each layer and the “norm” suffix indicates the value for the normal animals. The index takes a negative value when neurons in layer A of each animal show a shrinkage. When layer A1 was the recipient of the closed eye, the sign of the index was inversed. Thus, the shrinkage index represents the degree of soma size shrinkage in the closed-eye layer compared with the normal animals.