Size variations in synaptic terminals among different types of photoreceptors and across the zebrafish retina

Abstract

Photoreceptor synaptic terminals are responsible for transmitting visual information to downstream neurons. In vertebrate retinas, photoreceptor synaptic terminals are of different sizes and structures. The molecular mechanisms that underlie photoreceptor synaptic development are not clearly understood. Here, we have systematically examined the size variations in the synaptic terminals of cone and rod photoreceptors in the adult zebrafish retina. We reveal that the average cone pedicle sizes expand in the order of UV, blue, green, and red cones, echoing the increasing maximally sensitive wavelengths of the opsins expressed in the corresponding cone types. In addition, rod spherules are smaller than all cone pedicles. The terminals of each photoreceptor type also display distinct regional variations across the retina and between males and females. These findings establish the basis for using the zebrafish retina to study the molecular mechanisms that regulate the sizes and structures of photoreceptor terminals for proper visual functions.

Keywords: Pedicle; Photoreceptor; Ribbon synapse; Spherule; Synaptic terminal; Zebrafish.

Figure 1

The strategy for sampling local retinal regions. A. The diagram illustrates the dorsal, ventral, nasal, and temporal quadrants of the retina (divided by yellow lines). The vertical and horizontal line indicates the axes of the four quadrants where cryosections were collected for confocal microscopy. B. Each retinal cryosection was partitioned by a vertical line to exclude the developing marginal region (black) from the differentiated retina, which was further divided into five regions each with an angular subtense of 36 degrees. A 200-μm wide region (gray bars) in the center of each division was used to measure and quantify the sizes of photoreceptor terminals. Adapted and modified from Fu et al. (Fu et al., 2018).

Figure 2

Representative images of photoreceptor terminals in three EGFP transgenic zebrafish lines. A. Red, green, and blue (RGB) cones in the temporal peripheral retinal region are distinguished by immunostaining double cones with Zpr1 antibodies (red signals) in the genetic background of Tg(LCR^RH2-RH2–1:GFP)^pt112 transgenic fish (Fang et al., 2013). A’. An outer plexiform layer region in panel A is enlarged and displayed as single-channel images to illustrate the pedicles of red, green, and blue cone by arrow r, arrow g, and arrow b, respectively (Note the distinct red, yellow, and green signals). B. Arrows indicate the pedicles of UV cones in the temporal intermediate retinal region (revealed by EGFP expression in the Tg(SWS1:EGFP) background (Takechi et al., 2003). C. Arrows indicate rod spherules in the dorsal intermediate retinal region, highlighted by EGFP expression in the Tg(−3.7RHO:EGFP) background (Hamaoka et al., 2002). D. A representative serial optical sections through a UV cone pedicle. The middle section shows the widest span of the pedicle, which is defined as its diameter (double-headed arrow).

Figure 3

Cell-type-specific variations in terminal sizes of adult zebrafish retinal photoreceptors. A. A histogram illustrates that the terminal diameter averages (± standard deviations) are statistically different among red (R), green (G), blue (B), and UV cones as well as rods). One-way ANOVA test (F=5,810; p<0.0001). B. A pyramid grade illustrates the pair-wise comparisons of the means of terminal diameters by Tukey’s post hoc test revealed statistically significant differences in each pair (p<0.0001). The difference in mean diameters of each pair (base value minus column value) is noted in the corresponding grade. C. The table presents the differences in the average terminal sizes (μm) between females and males and the P-values (by Student’s t-test) according to photoreceptor types.

Figure 4

Regional variations of the photoreceptor terminal sizes. Histograms illustrate that the averages of photoreceptor terminal sizes of each of the five photoreceptor types are statistically different among nine retinal regions. The error bars represent standard deviations. Asterisks indicate the retinal regions that are significantly different from most of the other regions (see Supplementary tables 1–5 for all pair-wise comparisons by Tukey’s post hoc test). A. Red cones. F=15.73. B. Green cones. F=12.99. C. Blue cones. F=8.399. D. UV cones. F= 37.15. E. Rods. F=7.433. p<0.0001 for all one-way ANOVA tests.

Figure 5

Regional Variation Indices (RVIs), defined as 100 x {(average terminal diameter of local retinal region – average terminal diameter of whole retina) / average terminal diameter of whole retina}, of the synaptic terminals of five photoreceptor types. A. Red cones. B. Green cones. C. Blue cones. D. UV cones. E. Rods.