Ca2+ Dynamics in Retinal Horizontal Cells of Teleost Fish: Ca2+-Based Action Potentials and Tolerance to Hypoxia

Abstract

Horizontal cells (HCs) are retinal interneurons which provide feedback to photoreceptors to produce visual contrast. They are depolarized by glutamate released from photoreceptors, leading to a constant influx of Ca2+ which would be fatal to most neurons. In addition, HCs present spontaneous Ca2+-based action potentials, which are poorly understood and whose function is unknown. Given these unique Ca2+ dynamics, the present thesis sought to define action potentials (APs) and mechanisms of Ca2+ homeostasis in HCs. APs were observed in isolated goldfish HCs with electrophysiology, Ca2+ imaging, and voltage-sensitive dye imaging. Pharmacological inhibition of ion channels suggests APs required extracellular Ca2+ entry via L-type Ca2+ channels, followed by Ca2+-induced Ca2+ release from ryanodine receptors. Next, we developed a novel system to classify all four HC subtypes in vitro, and validated it with immunocytochemistry for a subtype-specific biomarker. All subtypes presented APs, although frequency and duration varied by subtype. APs were also found in HCs of tissue slices prepared from whole retina, where similar trends were found between subtype, frequency, and duration. This highlights subtype-specific differences in Ca2+ dynamics. Lastly, [Ca2+]i was monitored throughout hypoxia in HCs of the hypoxia-tolerant goldfish and the hypoxia-sensitive rainbow trout. In Ca2+ imaging experiments, hypoxia destabilized [Ca2+]i in HCs of trout; but in goldfish, HCs were resistant to the effects of hypoxia. However, when mitochondrial ATP-dependent K+ (mKATP) channels were inhibited, goldfish HCs lost the ability to maintain [Ca2+]i homeostasis during hypoxia. By contrast, in trout HCs, opening of mKATP stabilized [Ca2+]i during hypoxia. Furthermore, in goldfish, hypoxia protected against increases in [Ca2+]i caused by inhibiting glycolysis, showing that hypoxia is not just tolerated, but is actively protective in goldfish HCs. The present thesis includes the first comprehensive description of spontaneous Ca2+-based APs in HCs, and introduces the first cellular model of intrinsic hypoxic neuroprotection in the vertebrate retina.