Specificity of the horizontal cell-photoreceptor connections in the zebrafish (Danio rerio) retina

Abstract

Horizontal cells (HCs) are involved in establishing the center-surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)- and ultraviolet (UV)-sensitive single cones alternate with rows of red (R)- and green (G)-sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small-field (H1 and H2) and two large-field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1-GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI-labeled cone HC and connected to 50-200 rods.

Figure 1

Confocal image of DiI-labeled horizontal cells. Many horizontal cells are labeled up to several hundreds of micrometers away from the DiI insertion site.

Figure 2

The morphology of cone horizontal cells in the adult zebrafish retina. (A–C) The z-axis projections of serial confocal images of horizontal cells; (D–F) the single-plane images of these horizontal cells at their dendritic terminals. The H1 cell has a round soma, short dendrites, and dendritic terminal clusters that are localized in the center of the cell. The H2 cell has an irregular soma, more elongated dendrites and dendritic terminal clusters spread out throughout its dendritic field. The H3 cell is larger than the H1 and H2 cells and has long dendrites. The dendritic terminals of H3 cells consist of small clusters that form two overlaying rhombic patterns. H1, H2 and H3 cells all have an axon. The scale bar applies to all panels in the figure.

Figure 3

A rod horizontal cell in the retina of a 2-year-old zebrafish. (A) The z-axis projection of serial confocal images of the rod horizontal cell. A brightly DiI-stained horizontal cell axon (arrow) ends by the soma of this rod horizontal cell. (B) The axon (arrow) wraps around the soma of the rod horizontal cell (arrowheads). (C) The rod horizontal cell has about 160 individual spherical dendritic terminals, which arrange into single rows with seams between these single rows.

Figure 4

The cone connections of H1 cells. Selected images from a single confocal z-stack series. (A) Several H1 cells are visualized in a cluster. (B) The dendritic terminal rosettes of these H1 cells are organized into alternating double and single rows pattern. (C) Diagram of the idealized cone photoreceptor mosaic. The double cones (R and G) form the double rows and the alternating short- (UV) and long-single (B) cones form the single rows; the double rows alternate with the single rows; the neighboring single cones form a rhombus pattern (white rhombus). R, red-sensitive cones; G, green-sensitive cones; B, blue-sensitive cones; and UV, ultraviolet-sensitive cones. (D) The neighboring rosettes in the single rows of the H1 cell terminals form a rhombus pattern (blue rhombus). A magenta-green version of this figure is available as Supplemental Figure 1.

Figure 5

The cone connections of a H3 cell. Selected images from a single confocal z-stack series. (A) A single H3 cell and several H1 cells (H1s) are shown in this field. (B) The dendritic terminals of the H3 cell are organized into single rows that parallel the orientation of the double and single rows formed by the dendritic terminal rosettes of the H1 cells. (C) Diagram of the idealized cone photoreceptor mosaic. The neighboring single cones form overlapping rhombic patterns (white and brown rhombi). R, red-sensitive cones; G, green-sensitive cones; B, blue-sensitive cones; and UV, ultraviolet-sensitive cones. (D) The neighboring terminals of the H3 cell form overlapping rhombic patterns, marked in blue and purple. A magenta-green version of this figure is available as Supplemental Figure 2.

Figure 6

The cone connections of a H2 cell. (A) The z-axis projection of serial confocal images of a H2 cell and a H3 cell. (B) The dendritic terminals of the H2 and H3 cells. Some of the H2 cell dendritic terminal rosettes (in pairs) coincide with the H3 dendritic terminal pattern. (C) Diagram of the idealized cone photoreceptor mosaic. R, red-sensitive cones; G, green-sensitive cones; B, blue-sensitive cones; and UV, ultraviolet-sensitive cones. (D) The H2 cell appears to connect to G, B and UV cones (one of the two possible scenarios). The mosaic here is less regular than suggested in C; nevertheless, it is clear that UV cones (purple dots) are most often closer to G cones (green dots, blue lines) and B cones (blue dots) to the presumed R cones (open circles, open yellow lines). A magenta-green version of this figure is available as Supplemental Figure 3.

Figure 7

Confirming the cone connections of H1, H2 and H3 cells with SWS1-GFP zebrafish retinas. (A –C, and C′) A H1 cell; (D–F, and F′) a H2 cell; and (G–I, and I′) a H3 cell. (A, D and G) The z-axis projection of serial confocal images of the horizontal cell; (B, E and H) the dendritic terminals; (C, F and I) the overlay image of both the red (the horizontal cell dendritic terminals) and the green (GFP-positive UV cones) channels at the dendritic terminal level of the horizontal cell; (C′, F′ and I′) the enlarged view of the boxed area of a UV cone pedicle and the surrounding horizontal cell terminals in C, F and I, respectively. A magenta-green version of this figure is available as Supplemental Figure 4. The scale bar in panel A applies to panels A–I; the scale bar in panel C′ applies to panels C′, F′ and I′.

Figure 8

The frequency of the cone connections that were seen in the SWS1-GFP zebrafish retinas. R, red-sensitive cones; G, green-sensitive cones; B, blue-sensitive cones; and UV, ultraviolet-sensitive cones. Out of the H1 cells identified, 91% connected to the R, G and B cones; 9% may have connected to the UV cones as well. Out of the H2 cells identified, 68% connected to the G, B and UV cones; 29% connected to either R or G cones besides the B and UV cones; and 3% connected possibly to the R cones besides the B and UV cones. Out of the H3 cells identified, 96% connected to both the B and UV cones; 2% connected to the R and/or G cones besides the B and UV cones; and 2% connected to the UV cones and ambiguously to the B cones.

Figure 9

A model describing the cone horizontal cell pathways in zebrafish. Black arrows indicate major sign-conserving feedforward synapses whereas gray arrows indicate sign-inverting feedback synapses. R, red-sensitive cones; G, green-sensitive cones; B, blue-sensitive cones; and UV, ultraviolet-sensitive cones. Details in text.