Abstract
Purpose: :
Although photoreceptor cells in the vertebrate retina are anatomically connected or coupled by gap junctions, electrical coupling is thought to be weak. In goldfish, recent evidence indicates that rod-cone coupling is increased at night and that a circadian (24-hr) clock in the retina decreases coupling during the day by activating dopamine D2-like receptors (Ribelayga et al., 2007, ARVO). This study investigated whether the extent of coupling between photoreceptors in mice, rabbits and goldfish, as measured by intercellular propagation of Neurobiotin tracer after cut-loading, depends on the time of day.
Methods: :
Experiments were performed under dark-adapted conditions on goldfish, pigmented rabbits and CBA/CaJ mice. A razor blade was used to make several vertical cuts through the neural retina in the middle of the day or the middle of the night. The tissue was then incubated for 15 min in Ringer containing 0.05% Neurobiotin. Following cell loading and diffusion, the retina was washed, fixed and Neurobiotin visualized with streptavidin-conjugated Cy3 or Alexa-488 using a confocal microscope.
Results: :
Neurobiotin fluorescence was evident in photoreceptor cells along razor blade cuts both during the day and night in all three species (n = 3 for each condition and for each species). During the day, fluorescence was typically found in both rods and cones and restricted to the first 2-3 rows of photoreceptor cells along the cuts. In contrast, fluorescence in rods and cones extended far beyond the cuts at night, up to 200 µm from the cuts (> 20 rows of photoreceptors) in goldfish and up to 100 µm from the cuts (> 10 rows of photoreceptors) in mouse and rabbit. Finally, during the day, the dopamine D2-like antagonist spiperone (10 µM) increased photoreceptor coupling in all three species to the same extent as was observed at night under control conditions.
Conclusions: :
In mice, rabbits and fish under dark-adapted conditions, the extent of tracer coupling between photoreceptors was minimal during the day, but dramatically increased by more than 5-fold at night under control conditions and in the day in the presence of spiperone. These findings thus suggest that in mammals, as well as in fish, the retinal circadian clock controls rod-cone coupling by activating D2-like receptors in the day, so that rod-cone coupling is weak during the day, but remarkably robust at night. In addition, the similar day/night difference observed in goldfish with both tracer injections into single cones (Ribelayga et al., 2007) and with cut-loading substantiates the use of cut-loading as a technique to investigate the extent of photoreceptor coupling in the mammalian retina.
Keywords: gap junctions/coupling • circadian rhythms • photoreceptors