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S.J. Heflin, P.B. Cook; Immunocytochemical and Physiological Identification ofVoltage–gated Sodium Channels in Amacrine Cells ofTiger Salamander . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4270.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: It has been proposed that amacrine cells use action potentials to propagate their signal along their processes. Therefore, voltage–gated sodium channels should be present in the amacrine cell processes. This study attempts to locate voltage–gated sodium channels in tiger salamander retina, using both immunocytochemistry and physiological recordings. Methods: Salamander retinas were fixed with paraformaldehyde, cryoprotected and cut into 20 µm sections and mounted onto subbed slices. Immunocytochemistry (ICC) was performed using a pan–specific, monoclonal antibody (Sigma) raised against the intracellular loop III and IV of voltage–gated sodium channels. This region is conserved in all known vertebrates. Sections were subsequently incubated in a fluorescent secondary antibody and imaged using a confocal microscope. Salamander retinas were also dissected for live tissue physiology, mounted on membranes and cut to 250 micron slices. Physiology was performed on Lucifer yellow–filled cells, located on the IPL edge of the INL, that were most likely amacrine cells. Using a loose patch–clamp, both spontaneous and light–evoked action potentials were recorded. Following the physiological experiments, the slices were fixed with paraformaldehyde and processed for ICC following the section protocol with extended incubation times. Results: In retinal sections, fluorescent labeling was seen in IPL processes, and in the somatic plasma membrane and axons of the ganglion cells. Additional labeling was seen in unmylinated optic nerve fibers as the fibers exited the retina. Confocal imaging of the retinal slices revealed that the Lucifer yellow–filled amacrine cell processes ramified in the IPL. The IPL and ganglion cell somas and axonal processes of the slices were also positively labeled for voltage–gated sodium channels. Conclusions: Consistent immunostaining in both the retinal sections and slices indicate the presence of voltage–gated sodium channels in the amacrine cell processes and ganglion cell soma and axons. The physiological recordings of action potential spikes also indicate that the amacrine cells utilize the voltage–gated sodium channels for signal propagation.
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