Abstract
Abstract: :
Purpose: Regulation of cell shape and volume is essential to neuronal function. Fluctuations in the osmotic environment have the potential to disrupt the fidelity of neuronal signaling. Cell surface area regulation (SAR) is of particular importance because of its effect on membrane capacitance. Understanding the physiology of SAR will increase our knowledge of states of pathological membrane tension, e.g. acute angle-closure glaucoma. Methods: Individual horizontal cells were isolated from hybrid bass and skate retina and maintained in appropriate culture medium. For experiments on skate cells, solutions were modified as hypertonic or hypotonic to normal Ringer's solution (768 mOsmo) by altering the NaCl content +/- 20%. For hybrid bass cells, normal Ringer's (290 mOsmo) was supplemented with 12% sucrose to double the standard osmolarity. To measure intracellular calcium, cells were loaded with the calcium indicator dye Oregon Green (Molecular Probes) and analyzed on an inverted fluorescence microscope equipped with SlideBook (Intelligent Imaging Innovations). Results: Horizontal cells exhibit vacuole-like invaginations along their basal surface in response to a hypertonic shift in osmolarity. These vacuoles increase rapidly in size over 30 seconds. Upon return to normal Ringer's, the vacuoles slowly regress over 20 to 40 minutes. Vacuoles demonstrate "writhing" and in some cases form freely motile vesicles that migrate away from their site of formation. The sites of vacuole formation occur in stereotyped locations and are reproducible upon repeated stimulation. Replacing extracellular Ca++ with Mg++ does not affect vacuole formation. Intracellular calcium does not change during vacuole formation, but does increase during vacuole regression. To further understand the physiological consequences of SAR, we investigated glutamate induced calcium signals in horizontal cells. Whereas cells respond to repeated applications of 100 uM glutamate with an increase in intracellular Ca++ before osmotic stimulation, the cells are refractory to glutamate immediately following the application of a hypertonic stimulus. Conclusions: We demonstrated for the first time that retinal neurons form vacuole-like invaginations in response to osmotic stress, a phenomenon known to occur in many cell types throughout the CNS (Morris & Homann, 2001). Our findings suggest that SAR in retinal neurons may have a profound influence on neuronal signaling by attenuating the cell's response to glutamate and may possess a neuro-protective effect.
Keywords: horizontal cells • excitatory neurotransmitters • cell membrane/membrane specializations