Expression of neuronal connexin36 in AII amacrine cells of the mammalian retina

Abstract

We have studied the expression pattern of neuronal connexin36 (Cx36) in the mouse and rat retina. In vertical sections of both retinas, a polyclonal antibody directed against Cx36 produced punctate labeling in the inner plexiform layer (IPL). Intense immunoreactivity was localized to the entire OFF sublamina of the IPL, and much weaker staining could be observed in the ON sublamina. Double-labeling experiments in the rat retina with antibodies directed against parvalbumin indicate that Cx36 is expressed on dendrites of AII amacrine cells. Cx36-like immunoreactivity in sublamina a of the IPL did not overlap with lobular appendages or cell bodies of AII amacrine cells. In a mouse retinal slice preparation, AII amacrine and ON cone bipolar cells were intracellularly injected with Neurobiotin and counterstained with antibody against Cx36. Punctate labeling appeared to be in register with dendritic arborization of AII amacrines and cone bipolar cells in the ON sublamina of the IPL. Whereas AII amacrine cells isolated from the rat retina clearly displayed Cx36-like immunoreactivity, isolated ON cone bipolar cells were negative for Cx36. Axon terminals of rod bipolar cells were decorated with Cx36-positive contacts but did not express Cx36 themselves. These results indicate that Cx36 is expressed by AII amacrine cells in homologous and heterologous gap junctions made with AII amacrines and cone bipolar cells, respectively. The heterologous gap junctions appear to be heterotypic, because ON cone bipolar cells do not express Cx36.

Fig. 1.

Immunocytochemical localization of Cx36 in the retina of rat and mouse. A, B, Pattern of Cx36 immunoreactivity in vertical sections of the rat retina. The corresponding Nomarski image is shown in A.C, D, Pattern of Cx36 immunoreactivity in vertical sections of the mouse retina. The cytoplasmic staining of rod bipolar cell axons is attributable to paraformaldehyde fixation (see Materials and Methods). The corresponding Nomarski image is shown inC. Scale bars: A–D, 50 μm.

Fig. 2.

Expression of Cx36 in parvalbumin-immunoreactive cells in the rat retina.A, Double-labeling of a rat retina vertical section with antibodies against parvalbumin (red) and Cx36 (green). Only the vitreal half of the retina is shown. Arrows indicate lobular appendages of AII amacrine cells. B, Enlarged view of a single parvalbumin-positive AII amacrine cell from a different vertical section. All Cx36-immunoreactive puncta in sublamina b of the IPL are in register with AII amacrine cell dendrites. C, Horizontal section of the rat retina cut at the S3–S4 level of the IPL and double-labeled with parvalbumin (red) and Cx36 (green). Scale bars: A, 50 μm;B, C, 10 μm.

Fig. 3.

Cx36 immunoreactivity on injected and isolated AII amacrine cells. A, AII amacrine of the mouse retina filled with Neurobiotin and visualized with streptavidin–FITC. The picture represents a superimposed stack of 40 confocal images of 200 nm each. The stratification levels S1–S5 of the IPL are indicated ashorizontal lines to the left. The bistratified morphology of this cell type with lobular appendages in S1 (arrows) and a narrow-field dendritic tree in S3–S5 (arrowheads) is apparent. B, Pattern of Cx36 immunoreactivity superimposed on the cell shown inA. C, Single 200 nm confocal section of the same cell, superimposed on Cx36 staining pattern in the IPL. The cell body and most of the dendritic arborization are out of focus.D, E, Isolated AII amacrine cells from the rat retina double-labeled with antibodies against parvalbumin (red) and Cx36 (green). The cells display Cx36 immunoreactivity on their distal dendrites. Scale bars:A–E, 10 μm.

Fig. 4.

Injected and isolated ON cone bipolar cells do not display Cx36 immunoreactivity. A, ON cone bipolar cell of the mouse retina filled with Neurobiotin and visualized with streptavidin–FITC. The picture represents a superimposed stack of 34 confocal images of 200 nm each. B, Pattern of Cx36 immunoreactivity superimposed on the cell shown in A.C, Enlarged view of the axon terminal system of the same cell. Superimposed are 200 nm confocal sections of Neurobiotin and Cx36 staining. Cx36-immunoreactive puncta appear as cut vertically (arrows) or horizontally (arrowhead).D, Nomarski image of a cone bipolar cell isolated from the rat retina. E, The same cell shows immunoreactivity against recoverin and can therefore be identified as an ON cone bipolar cell. The numerous small, round cell bodies are photoreceptors that are also positive for recoverin. F, The same cell is not immunoreactive for Cx36. Scale bars: A–C, 10 μm;D–F, 10 μm.

Fig. 5.

Rod bipolar cells do not express Cx36.A, Double-labeling of a vertical section of rat retina with antibodies against PKC (green) and Cx36 (red). A region of the section with pronounced decoration of rod bipolar terminals with Cx36-positive puncta (arrows) is enlarged in the inset.B, Nomarski image of an isolated rod bipolar cell.C, Corresponding staining against PKC. D, The same cell is not immunoreactive for Cx36. Scale bars:A, 25 μm; inset in A, 10 μm; B–D, 10 μm.

Fig. 6.

Schematic drawing indicating the wiring of the mammalian retina in the IPL. AII amacrine cells make homotypic gap junctions containing Cx36 with other AII amacrines in the innermost layer of the IPL (depicted as Cx36–Cx36). In addition, heterotypic gap junctions with Cx36 expressed by AII amacrine cells (Cx36-?) are made with cone bipolar cells.C, Cones; R, rods; CB, cone bipolar cells; RB, rod bipolar cells;AII, AII amacrine cells; GC, ganglion cells.