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C. S. Miller, Jr., W. W. Kothmann, J. O'Brien; Bistratified Amacrine Cells in Zebrafish Show Lucifer Yellow Coupling. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3049.
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Connexin 35 (Cx35) forms gap junctions between many neurons in the retina and in the CNS. The degree of coupling mediated by these gap junctions is regulated by PKA phosphorylation, which previous work in our lab suggests is affected by light adaptation, likely in a cell-type specific manner, and possibly in a gap junction-specific manner. Our goal is to study these changes in more detail by identifying an amacrine cell type which is easy to find consistently, and whose gap junctions are mediated by Cx35.
Amacrine cells were impaled with microelectrodes filled with Neurobiotin and Lucifer Yellow in superfused flatmount retinas of light-adapted zebrafish (Danio rerio), and iontophoresed for 10 minutes with 3 Hz squarewaves of +/-1 nA . The retinas were fixed in 4% paraformaldehyde, and incubated with Cy3-conjugated streptavidin, a monoclonal antibody against Cx35, and a polyclonal antibody against Ser276-phosphorylated Cx35. Cell morphology, patterns of dye coupling, and phosphorylation state of Cx35 plaques were examined with a confocal microscope.
We are able to reliably impale a medium-field, bistratified amacrine cell whose soma is located within the IPL, but close to the INL. Dendrite thickness is 2-3 µm near the soma and about 0.5 µm at the termini. Dendrites exhibit few varicosities, a low degree of ramification, and cover an ellipsoid field. Maximal dendritic distance from the soma is about 100 µm. Arborizations occur at about 20% and 55% of the distance from the INL to the GCL.These cells form Cx35 gap junctions with one another and, in light-adapted retina, they are strongly tracer-coupled to each other, and less so to other types of amacrine cells. In addition, Lucifer Yellow is consistently observed to pass between neighboring cells of this type.
The ability to reliably impale a specific type of amacrine cell will greatly aid the detailed study of how light adaptation affects the phosphorylation state of Cx35 and electrical coupling in the retina. The unusual finding that Lucifer Yellow passes from one cell to another suggests that these cells form unusually large Cx35 junctions or are coupled by an additional connexin that has not yet been identified.
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