Abstract
Purpose: :
To generate a cone–specific connexin36 (Cx36) knockout (KO) mouse to study the role of Cx36–mediated rod–cone coupling in generating the rod–driven responses of ganglion cells.
Methods: :
A targeting construct that has the second exon of Cx36 flanked by loxP sites was used to generate homozygous Cx36Flx mice. Cone–specific Cx36KO animals were generated by crossing Cx36Flx mice with transgenic animals expressing Cre–recombinase driven by either the S (Blue, BP) or M (Red/Green, RGP) cone opsin promoter. Homozygous Cx36Flx/RGP–Cre positive animals and RGP–Cre negative littermate controls were analyzed by immunofluorescence and confocal microscopy. Light–evoked ganglion cell responses were obtained using extracellular recordings in a flattened retina–eyecup preparation.
Results: :
Cx36/RGP–Cre positive animals showed dramatically reduced (∼95%) staining for Cx36 in the outer plexiform layer (OPL) of the retina. The remaining label in the OPL was clustered and localized proximally. However, Cx36 staining within the inner plexiform layer (IPL) was unaffected when compared to that of homozygous Cx36Flx (Cre–negative) littermate controls and wild–type animals. In contrast, homozygous Cx36Flx/BP–Cre positive animals showed only a minor reduction of Cx36 label in the OPL, presumably due to the relatively small percentage of cones in the mouse retina that express the blue opsin. Electrophysiological recordings from ganglion cells in Cx36Flx/RGP–Cre positive mice and their Cre–negative littermates showed that the thresholds of the highest sensitivity responses were unaffected.
Conclusions: :
We successfully generated a cone–specific Cx36KO mouse. Our results indicate that the thresholds of high sensitivity ganglion cells are unaffected by disruption of Cx36–mediated rod–cone coupling. These data support the idea that the most sensitive rod–mediated signals are carried by the primary rod pathway and not the secondary rod–cone coupling pathway.
Keywords: gap junctions/coupling • ganglion cells • photoreceptors