Diffuse bipolar cells provide input to OFF parasol ganglion cells in the macaque retina

Abstract

Parasol retinal ganglion cells are more sensitive to luminance contrast and respond more transiently at all levels of adaptation than midget ganglion cells. This may be due, in part, to differences between bipolar cells that provide their input, and the goal of these experiments was to study these differences. Midget bipolar cells are known to be presynaptic to midget ganglion cells. To identify the bipolar cells presynaptic to parasol cells, these ganglion cells were intracellularly injected with Neurobiotin, cone bipolar cells were immunolabeled, and the double-labeled material was analyzed. In the electron microscope, we found that DB3 diffuse bipolar cells labeled by using antiserum to calbindin D-28k were presynaptic to OFF parasol cells. In the confocal microscope, DB3 bipolars costratified with OFF parasol cell dendrites and made significantly more appositions with them than expected due to chance. Flat midget bipolar cells were labeled with antiserum to recoverin. Although they made a few appositions with parasol cells, the number was no greater than would be expected when two sets of processes have overlapping distributions in the inner plexiform layer. DB2 diffuse bipolar cells were labeled with antibodies to excitatory amino acid transporter 2, and they also made appositions with OFF parasol cells. These results suggest that DB2 bipolar cells are also presynaptic to OFF parasol ganglion cells, but midget bipolar cells are not. We estimate that midperipheral OFF parasol cells receive approximately 500 synapses from 50 DB3 bipolar cells that, in turn, receive input from 250 cones.

Fig. 1

Western blot of tissue homogenates showing the proteins recognized by the anti-hEAAT2 serum. The blot shows protein extracts prepared from water-injected oocytes (lane 1) and hEAAT2-expressing oocytes (lane 2), 1.5 oocytes/lane; rat cortex (0.3 μg protein) (lane 3); monkey retina (6 μg protein) (lanes 4 and 5). The samples from monkey retina were stained with anti-hEAAT2 preincubated with either GST (lane 4) or GST-hEAAT2 (lane 5). hEAAT2 RNA-injected cells express a 73 kDA protein species that reacts with the antiserum. Although monkey retina shows two bands of ≈73 kDa and ≈37 kDa, both bands are effectively competed by preincubation with the hEAAT2-GST fusion protein.

Fig. 2

Camera lucida drawing of DB3 bipolar cell axons from a whole-mounted macaque retina. Individual DB3 axons terminals were difficult to distinguish, because neighboring axons often contacted each other (arrowheads). These are probably sites of gap junctions between DB3 cells. Second- and third-order axonal branch points typically occurred at varicosities (arrows). Scale bar = 100 μm.

Fig. 3

A DB3 bipolar cell axon (DB) is presynaptic to two OFF parasol ganglion cell dendrites (G) at ribbon synapses (arrowheads). The DB3 axons were labeled with anti-CaBP, and the parasol cell was filled with Neurobiotin. Scale bar = 0.5 μm.

Fig. 4

A DB3 bipolar cell (DB) makes a synapse (arrowhead) onto an OFF parasol ganglion cell dendrite (G). The HRP reaction product in the CaBP-IR DB3 axon creates a “fuzzy” appearance around synaptic vesicles. Note the unlabeled bipolar cell ribbon synapse in the upper right corner. Scale bar = 0.5 μm.

Fig. 5

In this double-labeled vertical section, CaBP-IR DB3 axons (green) ramify narrowly in the same layer of the IPL as dendrites from a filled OFF parasol cell (red, upper band). The lower red band contains dendrites from a neighboring ON parasol cell that was also filled. Also shown are a labeled DB3 cell body (upper right) and a filled parasol cell body (lower right). Scale bar = 10 μm.

Fig. 6

There were extensive appositions (arrows) between OFF parasol ganglion cell dendrites (red) and DB3 bipolar cell axons (green). This confocal micrograph represents a reconstructed stack of optical sections from the distal half of the IPL. Scale bar = 10 μm.

Fig. 7

In this control image for Figure 6, the OFF parasol cell dendrites (red channel) have been rotated 90° relative to the DB3 axons (green channel), which are in the same orientation as Figure 6. Apparent appositions between the two sets of processes (arrows) represent random interactions. Scale bar = 10 μm.

Fig. 8

Appositions (arrows) between OFF parasol ganglion cell dendrites (red) and FMB axons (green). Some apparent appositions (arrowhead) were separated along the z-axis and were therefore not counted. Scale bar = 10 μm.

Fig. 9

a: This triple-labeled vertical section contains hEAAT2-IR (red), recoverin-IR (green), and CaBP-IR (blue). In the IPL, anti-hEAAT2 labels the plasma membranes of bipolar cell axon varicosities. b: Some of these, mostly close to the top of the IPL, are FMB axons, which are also recoverin-IR throughout their cytoplasm (arrows). c: The other hEAAT-IR axon varicosities, which mostly lie below the FMB axons, are not recoverin-IR (arrowheads). This suggests that both FMB cells and DB2 bipolar cell axons are labeled with antisera to hEAAT2, but only FMB cells are labeled with antisera to recoverin. The CaBP-IR, DB3 axons (blue) are not hEAAT2-IR or recoverin-IR. Scale bar = 10 μm.

Fig. 10

Convergence onto a single OFF parasol cell in mid-peripheral retina. The parasol cell receives input from ≈50 DB3 bipolar cells and 250 cones. If each DB3 axonal varicosity that makes an apposition with an OFF parasol cell makes at least 1 synapse, then the 50 DB3 cells could provide 500 synapses onto the parasol cell.

Fig. 11

Summary diagram of proposed synapses from bipolar cells to parasol ganglion cells. Putative OFF cone bipolar cell types DB2 (DB2 BC) and DB3 (DB3 BC) receive mostly basal synapses from all of the cones in their dendritic field. DB3 bipolar cells make the majority of the bipolar cell synapses onto OFF parasol cells (OFF M GC), and they make homologous gap junctions with neighboring DB3 cells (crosshatched densities). DB2 bipolar cells provide the remainder of bipolar cell input to OFF parasol cells. C, cone; FMB, flat midget bipolar cell.