How Retinal Ganglion Cells Encode Anodal Break Excitation: The Role of T-type Ca2+ and Ih Channels

Abstract

Abstract: : Purpose:To determine the intrinsic membrane mechanisms shaping the voltage responses of ganglion cells on return from hyperpolarization. Methods:Whole cell recordings on ganglion cells were performed in the isolated whole mount Ambystoma tigrinum retina preparation. Recordings were performed in both voltage and current clamp configurations. Results:Current clamp recordings in normal Ringer's showed that ganglion cells demonstrate post anodal break excitation. Return from hyperpolarization reduced the latency to sodium spiking, in addition to generating a burst of spikes. Addition of tetrodotoxin unmasked a depolarizing voltage overshoot resulting from a preceding hyperpolarization. Thus the above effects are in part due to the superimposition of sodium spiking on this overshoot. Pharmacological analysis confirmed that the overshoot is composed of a low threshold Ca2+ spike mediated by T- type Ca2+ channels, and a depolarization from the non-selective cation current; Ih. The Ca2+ spike was eliminated on exposure to a low Ca2+ ringer containing Co2+. The Co2+ sensitive spike became regenerative at a very negative voltage range, in consonance with it being mediated by T-channels. It was also sensitive to low doses of the T-channel blocker, Mibefradil. With hyperpolarization, the Ih mediated volatge change involved a characteristic sag at the onset, and an overshoot on termination, of the hyperpolarizing pulse. Both these changes were sensitive to cesium, and were reduced on substitution of the bath Na+ by N-methyl D-glucamine. Corresponding pharmacological experiments performed in voltage clamp confirmed the presence of these two currents in these cells. Conclusions: The results demonstrate that hyperpolarization of retinal ganglion cells generates a burst of sodium spikes, in addition to reducing their latency to occurrence. The study identifies two intrinsic membrane currents, T-type Ca2+ and the Ih, as mediators of these phenomena. Hyperpolarization activates Ih, whereas it removes the resting level of inactivation from T-type channels. Both these effects contribute to the generation of a depolarizing voltage overshoot, with the resultant effects on Na+ action potential firing.

Keywords: ganglion cells • ion channels • electrophysiology: non-clinical