In their respective species, cell types DB1 and T1 had the highest axon stratification level and the second-largest axon arbor area, whereas types DB2 and T4 had the lowest stratification level and the second-smallest axon arbor area (Figure ?(Figure2;2; Table ?Table1).1). in the mouse retina made basal contacts with one or two of types, 3a, 3b, and 4. Approximately 3% of mouse rods were divergently connected to two OFF bipolar cells of different types, but macaque rods were solely connected to one OFF bipolar cell. Rod-rod gap junctions were localized at rod cell bodies and axons in the outer nuclear layer in both macaque and mouse retinas. The direct rod-OFF bipolar connection system is slightly more developed in the mouse retina than in the macaque retina, possibly as a fine-tuned adaptation to nocturnal conditions. This one-step direct synaptic pathway from rods to OFF bipolar cells may enhance the response speed to OFF light stimuli compared with more indirect pathways via rod-cone gap junctions (a two-step pathway) and via rod bipolar and AII amacrine cells (a three-step pathway). with 3% uranyl acetate in 80% methanol, dehydrated with ethanol, and embedded in araldite (Nisshin EM, Tokyo, Japan). A series of 817 radial sections 90 nm in thickness (73.5 m in total thickness) was taken from the block containing the Cloxiquine retina at 2.9C3.4 mm temporal to the foveal center. These sections were mounted on 120 formvar-coated single-slot grids and stained with 3% uranyl acetate in 80% methanol and Reynolds’ lead citrate. Electron micrographs of the series were acquired first at 400 using the JEM 1220 electron microscope (Jeol Ltd., Tokyo, Japan) at the Joint-Use Research Facilities of Hyogo College of Medicine. A total of 24 overlapping images were acquired from each individual section Cloxiquine at 3000, which captured a rectangular area of 90 187 m covering from the OPL to the ganglion cell layer (GCL) using a montage system of 4 6 negatives. These images were enlarged four-fold; thus, the final magnification of prints used for image analysis was 12000. This series was used for the examination of OFF bipolar cells and junctions between rod spherules. When the sites of candidate gap junctions were identified, additional electron micrographs were taken at 40000 with various tilting angles to reveal the characteristic structures of these gap junctions. Mice A series of 366 radial sections were prepared from the central area of the posterior retina of a C57BL/6J, 9-week-old, female mouse (20 g; SLC, Shizuoka, Japan), which is the same series that was used previously (Tsukamoto et al., 2001). This series was used for the examination of OFF bipolar cells and junctions between adjacent rod spherules. Another series of 133 tangential sections including the ONL were prepared from the posterior retina of a C57BL/6J, 8-week-old, male mouse (25 g; SLC, Shizuoka, Japan) for examination of rod-rod gap junctions in the ONL. This is different from the previous series of tangential sections used by Tsukamoto et al. (2001). Procedures for electron microscopy were similar to those described for the macaque retina above; these procedures were described in more detail by Tsukamoto et al. (2001). Examination area of the macaque retina The angular separation between the temporal edge of the optic disk and the foveal center is 15 in rhesus monkeys (and have similar gross retinal structures, the conversion of retinal distance to visual angle is 212 m/. The examination area was Cloxiquine located 3.00C3.25 mm temporal to the foveal center, and the center of this area was ~15 away from the foveal center. The top-view distribution of 3159 rod spherules and 237 cone pedicles (Figure ?(Figure1)1) was reconstructed from electron micrograph prints acquired at 4000 (10 enlargement of 400 negatives). This survey area, which formed a rough parallelogram of 73.5 224 m with an irregular contour located 3.00C3.25 mm temporal to the foveal center, was measured to be 0.01684 mm2 (Image-J; NIH, USA). Half of the cells that extended across the edge of the parallelogram were subtracted from the total number for density measurements. Thus, the corrected total numbers of rods and cones in this region were 2889 and 212, respectively, and the corresponding densities were 172 103 spherules/mm2 and 12.6 103 pedicles/mm2, respectively. The density ratio of rods to cones was 13.6. Open in a separate window Figure 1 Distribution of cone pedicles and rod spherules within the area of highest rod density in the macaque (and 0.05 were considered significant. Results Classification of OFF CD48 bipolar cells Nomenclature and quantitative assessment Side views of OFF (cone) bipolar cells are shown in Figure ?Figure2A2A for macaque and in Figure ?Figure2B2B for mouse. The nomenclature for the macaque OFF bipolar cell types (FMB, DB1, DB2, DB3a, and DB3b) follows.