Cone contacts, mosaics, and territories of bipolar cells in the mouse retina

Abstract

We report a quantitative analysis of the different bipolar cell types of the mouse retina. They were identified in wild-type mice by specific antibodies or in transgenic mouse lines by specific expression of green fluorescent protein or Clomeleon. The bipolar cell densities, their cone contacts, their dendritic coverage, and their axonal tiling were measured in retinal whole mounts. The results show that each and all cones are contacted by at least one member of any given type of bipolar cell (not considering genuine blue cones). Consequently, each cone feeds its light signals into a minimum of 10 different bipolar cells. Parallel processing of an image projected onto the retina, therefore, starts at the first synapse of the retina, the cone pedicle. The quantitative analysis suggests that our proposed catalog of 11 cone bipolar cells and one rod bipolar cell is complete, and all major bipolar cell types of the mouse retina appear to have been discovered.

Figure 1.

Bipolar cell types of the mouse retina (Ghosh et al., 2004) and the markers and transgenic mouse lines used in the present study. Csen, Calsenilin. For details, see Materials and Methods.

Figure 2.

Bipolar cells of the Gus–GFP transgenic mouse line. A, Vertical section through a Gus–GFP mouse retina immunostained for GFP (green) and calretinin (red). The retinal layers are indicated (OPL, INL, IPL, subdivided into 5 sublayers of equal thickness; GCL, ganglion cell layer). Scale bar, 25 μm. B, Horizontal view of a Type 7 bipolar cell dendritic tree. Four rod bipolar cells are more weakly labeled. C, Axon terminal of the cell in B. D, Axon terminals of rod bipolar cells.

Figure 3.

Cone contacts of Type 7 bipolar cells. A, C, Dendritic trees of two Type 7 bipolar cells. B, D, Their cone contacts (red label, GluR5 marking cone pedicles). E, Frequency histogram of the number of cone contacts of individual Type 7 cells. F, Group of neighboring Type 7 cells. G, Same field as in F (red label, glypho-stained cone pedicles). H, Blue circles mark cones contacted by two bipolar cells, and white circles mark cones contacted by one bipolar cell. I, Dendritic fields of Type 7 bipolar cells outlined by white lines. J, Axon terminals of the bipolar cells in I, outlined by the white lines. Scale bar: A–D, 12.5 μm; F–H, 15 μm; I, J, 20 μm.

Figure 4.

Retinal distribution and cone contacts of Type 3a and Type 3b bipolar cells. A, Dendritic trees of Type 3a bipolar cells immunostained for HCN4 (green), cone pedicles are marked by GluR5 (red). B, Axon terminals of the Type 3a bipolar cells in A. C, Frequency histogram of the number of cone contacts of Type 3a cells. D, Dendritic trees of Type 3b bipolar cells immunostained for PKARIIβ (green), cone pedicles are marked by GluR5 (red). E, Axon terminals of the Type 3b bipolar cells in D. F, Frequency histogram of the number of cone contacts of Type 3a and Type 3b cells. Scale bar, 25 μm.

Figure 5.

Dendritic trees of bipolar cells in a retinal whole mount that was triple labeled for calsenilin (Csen), NK₃R, and GluR5. A, Dendritic trees of Type 4 bipolar cells immunolabeled for calsenilin (green), cone pedicles are marked by GluR5 (red). B, Dendritic trees of Type 1 and Type 2 bipolar cells immunolabeled for NK₃R (green), cone pedicles are marked by GluR5 (red). Scale bar, 25 μm.

Figure 6.

Syt2 immunostaining of Type 2 and Type 6 bipolar cells. A, Vertical section, double labeled for Syt2 (green) and calretinin (red). Axon terminals of Type 2 cells (in sublamina 1 and 2) are strongly labeled, and those of Type 6 cells (in sublamina 3–5) are weakly labeled. B, Axon terminals of Type 2 cells immunostained in a retinal whole mount for Syt2. C, Axon terminals of Type 6 cells. Scale bar, 25 μm.

Figure 7.

Analysis of the bipolar cells immunoreactive for Syt2. A–C, Vertical section through the retina of a Gus–GFP mouse. A, Syt2 immunoreactivity (red) in axon terminals of Type 2 and Type 6 bipolar cells. B, GFP labeling (green) of Type 7 and rod bipolar cell axon terminals. C, Superposition of A and B shows no colocalization. D–F, Vertical section through the retina of a Clm12 mouse. D, Syt2 immunoreactivity (red). E, GFP labeling (green). F, Superposition of D and E shows that all Syt2-immunoreactive bipolar cells in D express also Clomeleon. Scale bar, 25 μm.

Figure 8.

Dendritic network and cone contacts of Type 2 bipolar cells. A, Dendritic network of Type 2 bipolar cells in the Clm12 mouse (green) and cone pedicles (PNA labeled, red). B, Frequency histogram of the number of cone contacts of Type 2 bipolar cells. C, Dendritic trees of the Type 2 bipolar cells in A. D, Overlap of the dendritic trees in C. Scale bar: A, 25 μm; C, D, 22 μm.

Figure 9.

Whole mount of a Clm1 mouse retina, triple labeled for NK₃R, Syt2, and GFP. A, NK₃R-labeled bipolar cell perikarya (red). B, Same field as in A; Syt2 labeling of Type 2 cell bodies (green). C, Superposition of A and B shows that Type 2 cells are double labeled for NK₃R and Syt2. Bipolar cells expressing only NK₃R represent Type 1 cells. D, Same field as in A double labeled for NK₃R (red) and Clm (GFP, blue). With one exception (arrowhead), all GFP-labeled, blue cell bodies express also NK₃R (red). E, Same field as in A double labeled for Syt2 (green) and Clm (GFP, blue). All GFP-labeled, blue cell bodies do not express Syt2 (green). F, Same field as in A, showing the dendritic fields of the Clm (GFP, blue)-labeled bipolar cells. The two bipolar cells marked by the arrows are not immunoreactive for Syt2 in B and E but are immunoreactive for NK₃R in A and D and thus represent Type 1 bipolar cells. Their dendritic fields are outlined. Scale bar, 25 μm.

Figure 10.

Type 5 bipolar cells are labeled in the 5-HT₃R–EGFP mouse retina. A, Vertical section through the retina of the 5-HT₃R–EGFP mouse. B, Vertical section double labeled for GFP (green) and calretinin (red). C, D, Same field of a whole mount of a 5-HT₃R–EGFP retina. C, Focus on the cell bodies of GFP-labeled bipolar cells. D, Focus on the cone pedicles marked by GluR5 immunostaining (red). Scale bar: A, B, 25 μm; C, D, 32 μm.

Figure 11.

Summary diagram of the bipolar cell types as defined in the present study. A total of 11 cone bipolar cells and one rod bipolar cell could be distinguished by immunocytochemical staining (outlines) or by GFP/Clomeleon expression in transgenic mouse lines (green cells).