Abstract
Purpose :
Ca2+ regulates distinct cellular processes in cone photoreceptors (PRs) from photoresponse recovery in the outer segment (OS) to neurotransmission in the synapse. Mitochondria are located directly between the OS and the cell body and are known to buffer Ca2+ in other cell types. We hypothesize that mitochondria buffer Ca2+ within cone PRs to compartmentalize Ca2+ signaling.
Methods :
Adult zebrafish retinas expressing a genetically encoded fluorescent Ca2+ sensor in the cone PR cytosol (GCaMP3) or mitochondria (mitoGCaMP3), were dissected, flat-mounted, stained with BODIPY, and sliced into 400-µm sections. Slices were transferred to an imaging chamber. Slices were perfused with Ringer’s solution, then treated with either increased extracellular Ca2+, 25 μM sildenafil, 10 mM KCl, or with 10 μM Ru360. GCaMP fluorescence was monitored by confocal microscopy.
Results :
We found that synaptic and cell body Ca2+ levels can be selectively increased when the cells are depolarized with KCl, whereas OS Ca2+ levels remain unchanged. Conversely, OS Ca2+ can be dramatically increased using a Pde6 inhibitor sildenafil. Treatment with sildenafil for 30 minutes causes Ca2+ to accumulate in the OS, while Ca2+ pools in the cell body and synapse are low. These findings indicate that Ca2+ diffusion between PR compartments is controlled. To determine if mitochondria maintain distinct Ca2+ pools, we monitored mitochondrial Ca2+ levels using mitoGCaMP3. Raising Ca2+ either in the OS or cell body causes an apparent rise in mitochondrial Ca2+. Treatment of slices with Ru360, an inhibitor of the mitochondrial calcium uniporter (MCU), abolishes compartmentalization, implicating mitochondria in the segregation of Ca2+ pools.
Conclusions :
PRs use Ca2+ to modulate signaling processes in the OS, cell body, and synapse. Here we show that Ca2+ is compartmentalized between OS and cell body in PRs, and this gradient is maintained by mitochondrial Ca2+ buffering via the MCU. The influence of OS Ca2+ and cell body Ca2+ on mitochondrial function are not yet known. Understanding how Ca2+ homeostasis is regulated in PRs will provide an understanding for how cells in the retina stay healthy and how and mitochondria respond to energy demands.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.