Rod and Cone Connections With Bipolar Cells in the Rabbit Retina

Abstract

Rod and cone pathways are segregated in the first stage of the retina: cones synapse with both ON- and OFF-cone bipolar cells while rods contact only rod bipolar cells. However, there is an exception to this specific wiring in that rods also contact certain OFF cone bipolar cells, providing a tertiary rod pathway. Recently, it has been proposed that there is even more crossover between rod and cone pathways. Physiological recordings suggested that rod bipolar cells receive input from cones, and ON cone bipolar cells can receive input from rods, in addition to the established pathways. To image their rod and cone contacts, we have dye-filled individual rod bipolar cells in the rabbit retina. We report that approximately half the rod bipolar cells receive one or two cone contacts. Dye-filling AII amacrine cells, combined with subtractive labeling, revealed most of the ON cone bipolar cells to which they were coupled, including the occasional blue cone bipolar cell, identified by its contacts with blue cones. Imaging the AII-coupled ON cone bipolar dendrites in this way showed that they contact cones exclusively. We conclude that there is some limited cone input to rod bipolar cells, but we could find no evidence for rod contacts with ON cone bipolar cells. The tertiary rod OFF pathway operates via direct contacts between rods and OFF cone bipolar cells. In contrast, our results do not support the presence of a tertiary rod ON pathway in the rabbit retina.

Keywords: AII amacrine cell; bipolar cell; cone; retina; rod.

Figure 1

Rod and cone mosaic. (A) mGluR6 labeled puncta within the OPL of the rabbit retina reveal two separate labeling patterns. Large doublets, arrowheads, which correspond to the tips of two rod bipolar dendrites at each rod spherule and smaller clustered puncta which correspond to cone pedicles (arrow). (B) Addition of GluR5 labeling reveal the locations of cones. (C) Cluster analysis of mGluR6 puncta reveal that two distinct populations are readily statistically separable, ellipses show 95% confidence limits. (D) Mean volume and mean intensity of mGluR6 labeling (arbitrary units), (mean + SD, n = 3, p < 0.05).

Figure 2

Rod and cone mosaic. (A) mGluR6 labeling in the OPL shows clusters of fine terminals, associated with cone pedicles (circled) and larger, brighter doublets, associated with rod spherules. (B) Same field labeled with an antibody against ribeye to stain synaptic ribbons shows a cluster of small ribbons associated with cone pedicles (circled) and larger horseshoe-shaped ribbons at rod spherules. (C) Triple label, same field, shows GluR5 labeling at cone pedicles (circled) with the smaller ribbons and mGluR6 clusters. At rod spherules, the curved synaptic ribbon encloses the mGluR6 doublets.

Figure 3

PKC labeling, rabbit retina, wholemount. (A) The cell bodies of rod bipolar cells are stained for PKC. (B) Focus in the OPL shows a large population of very fine processes which terminate at rod spherules.

Figure 4

Triple label showing rod and occasional cone contacts of PKC labeled rod bipolar cells. RBCs receive putative contact from cones. (A) Labeling of rod bipolar cell dendrites at the level of the OPL with an antibody against PKC-α demonstrates that many mGluR6 clusters at rod spherules are double-labeled. Cone pedicles are shown by the combination of fine mGluR6 terminals and GluR5 labeling. Some cone pedicles, circled, receive dendritic contacts from PKC labeled rod bipolar cells. (B–D) enlarged view of the square area in (A) to show individual channels. (B) A very fine branch from a rod bipolar dendrite reaches out to a cone pedicle. (C) The rod bipolar dendrite contacts the cone pedicle labeled for GluR5. (D) The rod bipolar dendrite is double-labeled for mGluR6 where it terminates within the cone pedicle. Note: some horizontal banding in this image resulted from saturation of the RBC cell bodies, which were saturated because we set the gain to view the fine dendrites; it did not affect the analysis of connectivity.

Figure 5

Rod bipolar cell—dye fills. (A) A single rod bipolar cell filled with Neurobiotin (green), focus on the dendrites in the OPL. The cell body is blurred because it is out of focus. This is a perfect fill which provides complete details of the dendritic tree. The major dendritic branches terminate at approximately 100 rod spherules. (B) Enlarged view of square in (A) to show two cone pedicles labeled for GluR5 (blue). The left cone pedicle, circled, receives a branch from the dye-filled rod bipolar cell. (C) Enlarged view of square in (A) to show the rod bipolar cell contact with a cone terminates at a fine mGluR6 (red) puncta within the cone pedicle, circled. On the right side, the rod bipolar cell dendrites appear to approach another cone pedicle but terminate at two close-by rod spherules. This can be seen because the rod bipolar dendrites terminate at one half of an mGluR6 doublet, arrow. This pattern is seen throughout the dendritic tree because normally rod spherules receive input from two different rod bipolar cells. (D–F) An example from another Neurobiotin filled rod bipolar cell where a rod bipolar dendrite reaches to the center of a cone pedicle, circled. Again, except for the cone contact, the rod bipolar dendrites terminate at half an mGluR6 doublet marking the site of rod spherules.

Figure 6

(A) A single dye filled rod bipolar cell (green) with cone contacts against a background of PKC labeled rod bipolar cells (red). Focus on the rod bipolar terminal deep in the IPL. (B) The axon terminal field of the filled rod bipolar cell (black) against a background showing the terminal fields of all PKC-labeled rod bipolar cells. The filled cell appears to be part of a single mosaic with a coverage of 0.75, typical of bipolar cells.

Figure 7

(A) Vertical section through a patch of dye-filled AII amacrine cells (green), which have stout descending dendrites and tethered lobules in sublamina a, and a dense plexus in sublamina b, beneath the lower cholinergic band (magenta), used as a depth marker. Above the AII amacrine cells, there are numerous tracer-coupled cone bipolar cells (also green), with smaller somas and axons which descend and become lost in the AII plexus. Z-axis projection, thickness, 18 μm. (B) Wholemount view of dye-filled AII amacrine cells showing typical morphology with tethered lobules, projection, thickness 10 μm. (C) Same field, focus in the OPL showing tracer-coupled ON cone bipolar cells with dendrites, projection, thickness 10 μm. (D–G) Subtractive labeling. (D) Section through a dye-filled AII patch, with overlying dye-coupled ON cone bipolar cells. (E) Same field, calretinin labeled AII amacrine cells (magenta). (F) Same field, double label showing dye-filled AII amacrine cells (Neurobiotin, green + calretinin, magenta = white). In the background there are other AII amacrine cells outside the dye-coupled patch, which are stained only for calretinin (magenta). Above the AII amacrine cells, there are dye-coupled ON cone bipolar cells (Neurobiotin only, green). (G) Subtracting the AII amacrine cells (magenta) isolates the cone bipolar cells (green) and reveals bipolar cells axons which descend to sublamina b of the IPL. Terminals at different depths show the presence of several different ON cone bipolar types. (D–G) Z-axis projection, thickness 15 μm.

Figure 8

(A) A Neurobiotin-filled patch, following the dye injection of a single AII amacrine cell, shows a large number of dye-coupled ON cone bipolar cells (green) with dendrites in the OPL, projection, thickness 12 μm. (B) Labeling the rod-cone mosaic for mGluR6 (red: rod bipolar tips at rod spherules) and GluR5 (blue, cone pedicles) shows that the dye-coupled ON cone bipolar dendrites only contact cones, projection, thickness 12 μm. (C) Z-axis rotation to show a section through the same dye-coupled patch shows that most rod spherules lie substantially above both the cone pedicles and the dendrites of ON cone bipolar cells, projection, thickness 22 μm.

Figure 9

ON cone bipolar cells contact cones exclusively. (A) The rod-cone mosaic labeled for mGluR6 (rod spherules, red) and GluR5 (cone pedicles , blue), projection, thickness 8 μm. (B) Same field showing detail of dye-coupled ON cone bipolar cells (green) with dendrites reaching out to GluR5 labeled cone pedicles (blue), projection, thickness 8 μm. (C) There is no indication that ON cone bipolar cell dendrites terminate at mGluR6 labeled rod [spherules (red), projection, thickness 8 μm. A green dendrite passes underneath two rod spherules (white arrowheads). (D) Z-axis rotation shows that the bipolar dendrite is at a different depth than the two rod spherules (white arrows) and simply passes underneath without contact, projection, sliced in Imaris, thickness 2 μm.

Figure 10

Blue cone bipolar cells contact blue cones exclusively. (A,C) Two separate blue cone bipolar cells, both dye coupled to an injected AII patch. They are identified as blue cone bipolar cells by their long eccentric dendrites which bypass other cones to terminate at blue cones. (B,D) Same fields, with blue cones circled, with weak GluR5 labeling and prominent mGluR6 terminals. All panels, projection, thickness 10 μm.