Abstract
Purpose :
Current knowledge of the rhythmicity of photoreceptor outer segment phagocytosis by the retinal pigment epithelium (RPE) is mostly obtained from rod-dominant animals, such as mice. Most of the studies report the burst of rod phagocytosis to occur after light onset in the morning. However, for cones either one peak after light offset or two peaks after light onset and offset have been demonstrated depending on the animal species. Moreover, it is not known if the rhythmicity of phagocytosis varies between the different cone types. We investigated the rhythmicity of phagocytosis of rods and all cone types (red, green, blue, UV) individually by using cone-dominant larval zebrafish. Furthermore, we tested whether the rhythmicity was retained in constant darkness.
Methods :
Histological sections of whole eyes were prepared from 7 days old zebrafish that were euthanized at 7 time points during 24 h both under normal light cycle (14/10 h of light/dark) and in constant darkness. The sections were immunolabelled against proteins specific for RPE and the different photoreceptor types, and then imaged with a confocal microscope. The number of phagocytosed outer segment particles for each photoreceptor type was quantified from the images (≥3 fish, n=5-10 eyes/time point). These numbers were then compared at distinct time points to determine the level of phagocytosis during the 24 h cycle at both light conditions.
Results :
Phagocytosis of all cone types showed rhythmic nature in zebrafish larvae with one peak emerging after light onset in the morning and one after light offset in the evening. During the peaks, the number of phagocytosed outer segment particles was significantly higher compared to the basal level with the largest difference seen in red cones (morning: p<0,0001, evening: p<0,0002). Interestingly, the evening phagocytosis peak seems to last longer than the morning peak for all cone types. Rod phagocytosis, instead, showed only one slight peak in the morning compared to the basal level (p>0,07). Our data also indicates that the peaks persist in constant darkness.
Conclusions :
Our results show that phagocytosis of all the different cones peaks both after light onset and offset in zebrafish larvae. Rod phagocytosis, instead, shows only weak rhythmicity which might be due to rods not being fully developed. Our work strengthens the recent evidence that cone phagocytosis peaks twice during the circadian cycle.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.