Abstract
Abstract: :
Purpose: Neurons possess elegant mechanisms to regulate their volume and measure their shape, crucial factors for maintaining proper synaptic transmission. The distinct morphology of neurons in the outer retina provides an ideal model to reveal the molecular mechanisms of volume regulation. Previously, we have shown that retinal neurons form numerous vacuole–like dilations (VLDs) in response to hyperosmotic stimuli. In this study we have examined the molecular basis of these regulatory programs. Methods: Individual retinal neurons were isolated from hybrid bass and perch retina and maintained in culture for 3 days. Normal Ringer's solution (325 mOsmo) was supplemented with sucrose (or glucose) to increase the standard osmolarity. Cell membranes were labeled with the fluorescent dye CellTracker CM–DiI (Molecular Probes; 1µM for 15 minutes) and analyzed on a SB2–AOBS confocal microscope (Leica). Results: Horizontal and bipolar cells are very sensitive to changes in the osmotic environment and form VLDs with increments in osmolarity of as little as 12%. VLDs form from surface membrane and require cell attachment, either to neighboring cells or the substratum. Non–adherent cells are unable to form VLDs and undergo significant volume changes (decreasing by around 15% with a 50% increase in osmolarity), whereas the dimensions of adherent cells are largely unaffected. VLDs form in reproducible locations upon repeated stimulation. To further understand the mechanism of VLD formation, we investigated agents known to disrupt the dynamics of the actin cytoskeleton. Neither phalloidin (stabilization, 1µM) nor cytochalsin B (depolymerization, 1µM) had any effect on osmotically driven VLD formation or regression. Furthermore, when cells were treated with the metabolic inhibitor, sodium cyanide, the process of VLD formation and regression was unaffected, though this treatment alone induced small intracellular vesicles. Interestingly, hypertonic treatment of retinal slice does not cause significant changes in the shape or volume of cells in the inner nuclear layer, suggesting that similar mechanisms of volume regulation also occur in the intact retina. Conclusions: Our results indicate that horizontal and bipolar cells preserve their shape and volume in part by forming VLDs in response to osmotic stress. The formation and regression of VLDs does not depend on actin cytoskeleton or cellular energetics. The stereotyped locations of vacuole formation suggest that the cell has preferred sites for VLD formation, perhaps to avoid disrupting tightly coupled areas in order to preserve synaptic communication.
Keywords: cell membrane/membrane specializations • horizontal cells • retina: distal (photoreceptors, horizontal cells, bipolar cells)