Studies on the survival of central nervous system of hypoxia-tolerant species under challenges of reduced energy availability have characterised adaptive mechanisms of brain at the cell and tissue level that lead to reduced excitability and protection. However, evidence of hypoxic suppression of retinal activity in these species has not been followed up with mechanistic studies. Microspectrofluorometric monitoring of intracellular free Ca2+ concentration ([Ca2+]i) is useful for identifying cellular mechanisms that may lead to adaptive strategies, as unregulated increases in [Ca2+]i cause toxicity. Horizontal cells (HCs) are second order retinal neurons that receive tonic excitatory input from photoreceptors, and possess voltage-gated Ca2+ conductances and other channels that can facilitate toxic increases in [Ca2+]i under conditions of reduced energy availability (modeled as oxygen-glucose deprivation, OGD). It was demonstrated that isolated HCs of the hypoxia-tolerant goldfish display spontaneous, transient [Ca2+]i activity (SA) which decreased in amplitude and area under the curve following OGD or glucose removal (20 min) without recovery. SA was shown to be dependent on extracellular Ca2+ influx through voltage-gated Ca2+ channels, though mechanisms of SA generation and regulation has yet to be determined. Additionally, glutamate-elicited peak increases in [Ca2+]i were reduced after 20 min of OGD. The removal of O2 during OGD insult seemed to be protective as an increase in baseline [Ca2+]i was seen during and following glucose removal under normoxic conditions. The mechanisms mediating these decreases in spontaneous and elicited [Ca2+]i activity are currently unknown, though candidate pathways are discussed. This thesis contributes a hint towards how HCs may tolerate conditions of low energy availability, which may also inform investigations on their role in situ during these insults.

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.