Abstract
Abstract: :
Purpose: Various aspects of photoreceptor function are dynamically regulated. Both synaptic ribbon length and visual threshold change in a 24h light/dark cycle in the mouse retina. We tested if there was a causal link between light activation and ribbon length and between ribbon length and visual threshold. That is we asked: Is synaptic ribbon length governed via circadian factors or factors relating to the phototransduction cascade? Methods: Visual thresholds were measured in a water–maze testing C57BL/6J + vs. c–2J. Threshold was measured every 4 hours over a 12h/12h L/D cycle and throughout 24 h of constant darkness. Mice for synaptic ribbon measurements were anesthetized with 0.3cc of 25% pentobarbital IP. The mice were perfused through the heart with saline followed by fixation with 1% PLP (0.5% Na periodate, 0.34% lysine, and 1% paraformaldehyde) for 3–5 minutes. Eyecups were made, then embedded in O.C.T. and frozen sectioned at 7um. Sections were incubated with B16 primary antibody and Cy3 secondary antibody to view synaptic ribbons with fluorescence microscopy. Synaptic ribbon lengths were measured every four hours over the 48 hour period. Results: We found that visual thresholds were elevated in all mice maintained in cycling light when compared to those in constant darkness. The cycle–locked changes in visual threshold continued in both strains throughout the 24h constant dark period. Similarly synaptic ribbon length in both strains continued their cycle locked changes in length throughout the 24h constant dark–period. As both ribbon length and threshold showed the same phase lock, we are interested in determining if the ribbons change in length is related to the phototransduction cascade in the photoreceptor. In the vertebrate retina, guanylate cyclase regulates cGMP production. When cGMP levels are high Na+ channels remain open resulting in a depolarization of photoreceptor cells, as cGMP drop Na+ channels close resulting in hyperpolarization of the photoreceptor. Thus, we are manipulating directly cGMP levels and assessing the effects on both ribbon length and visual threshold. Conclusions: Our results indicate that both visual thresholds and synaptic ribbon length change over the circadian cycle and are not strictly dependant on ambient light. We hypothisize that ribbon length and phototransduction are couple.
Keywords: circadian rhythms • photoreceptors • retina: distal (photoreceptors, horizontal cells, bipolar cells)