Abstract
Purpose::
In the rabbit retina, there are two types of horizontal cell (HC). A-type HCs are axonless and they are extensively coupled via Cx50 gap junctions. B-type HCs are axon bearing; the somatic dendrites receive input from cones and form a second coupled network while B-type axon terminals (ATs) receive input from rods and form a third network in the outer plexiform layer (OPL). We wanted to determine which connexins are expressed by B-type horizontal cells. The mouse retina has only one type of axon bearing HC which has been shown to express Cx57 (Hombach et al., 2004).
Methods::
HCs were dye injected with Lucifer Yellow and Neurobiotin in DAPI-incubated isolated rabbit retina. Tissues were then immunolabeled with antibodies against Cx 57. The relationship of Cx57 to the B-type horizontal cell dendritic tips was analyzed by confocal microscopy
Results::
Cx57 from the rabbit retina was sequenced by PCR. A peptide from the C-terminus was used to raise a monoclonal antibody which recognized a Cx57 fusion protein in Western blots. In vertical sections of the retina, the Cx57 antibody produced a narrow band of small plaques in the OPL consistent with the placement of HCs. The positions of rod and cone terminals were marked with mGluR6 or ribeye. The Cx57 labeling occurred below the photoreceptor terminals and showed no association with the rod or cone terminals. The three horizontal cell matrices in the OPL could be independently filled with Neurobiotin. Cx57 plaques were not present in A-type HCs. Surprisingly, we could find no specific Cx57 labeling in the matrix of B-type soma dendrites. The B-type axon terminal matrix was very dense with an estimated coverage of 10 and it was well labeled with Cx57 plaques. High resolution images showed that Cx57 plaques occurred at crossovers or contacts in the AT matrix.
Conclusions::
We report that Cx57 immunoreactivity is present in the B-type HC-AT matrix. This confirms that different horizontal cell types use different connexins in the rabbit retina. The connexin responsible for coupling in the somatic dendrites of B-type HCs is still unknown.
Keywords: horizontal cells • gap junctions/coupling • retina: distal (photoreceptors, horizontal cells, bipolar cells)