Abstract
Purpose:
The nuclei of rod and cone photoreceptors form the outer nuclear layer (ONL) of the retina, with cone nuclei localized specifically at the apical side. The expression of EGFP-Kash2 in cones disrupts this organization and results in the mislocalization of the cone nuclei to the basal side of the ONL. We examined how the localization of cone nuclei affects the function of cones by using transgenic EGFP-Kash2 mice.
Methods:
EGFP-Kash2 transgenic mice were crossed with HGRP-Cre mice to induce cone-specific expression of EGFP-Kash2 and immunostaining was performed to confirm cone nuclei mispositioning and synaptic defects. To facilitate cone recordings, all mice were in the Gnat1-/- (transducin α-subunit knockout) background and were dark adapted overnight prior to the recordings. Cone photoresponses were obtained from transretinal recordings, and cone b-wave responses were obtained from in-vivo ERG recordings. We determined the functional properties of cones in dark-adapted conditions and their recovery from exposure to bright bleaching light.
Results:
EGFP-Kash2 expression in cones resulted in the mislocalization of >95% of cone nuclei to the basal side of the ONL as well as the OPL. While this mislocalization did not affect the inner and outer segments in 6 month-old mice, the structural organization of cone pedicles was severely disrupted. We carried out electrophysiological experiments to determine the functional consequences of these observations. In dark-adapted conditions, the mislocalization of cone nuclei did not affect the kinetics or maximal amplitude of cone flash responses, but decreased the sensitivity (I1/2) by 2.8-fold. In addition, the maximal cone b-wave amplitude was decreased by 1.6-fold in EGFP-Kash2/HGRP-Cre mice. Notably, the level of cone b-wave sensitivity recovery 50 minutes following a bright bleach in-vivo was 2.6-fold lower. The level of cone sensitivity recovery 7 minutes after a bleach in isolated retina was also suppressed by 1.5-fold.
Conclusions:
The mislocalization of cone nuclei reduced the cone sensitivity and compromised the synaptic transmission between cones and bipolar cells in EGFP-Kash2/HGRP-Cre mice. Notably, cone dark adaptation was impaired both in vivo and in isolated retina suggesting that the positioning of cone nuclei is important in maintaining the chromophore supply to cones through both the pigment epithelium and the retina visual cycles.
Keywords: 508 electrophysiology: non-clinical •
648 photoreceptors •
688 retina