Mammalian neurons rapidly undergo excitotoxic cell death during anoxia, while neurons from the anoxia-tolerant painted turtle can survive without oxygen for hours without apparent damage. An anoxia-mediated decrease in AMPA receptor currents are an important part of the turtle’s natural defence however the mechanism underlying it is unknown. Here I investigate a mechanism that involves activation of a mitochondrial KATP channel that subsequently signals a decrease in AMPAR currents. Whole-cell AMPAR currents were stable during normoxia, but anoxia or pharmacological activation of mKATP channels resulted in a 50% decrease in AMPAR currents. Conversely, mKATP antagonists blocked the anoxia-mediated decrease. Mitochondrial KCa channel modulators responded similarly. Blocking the Ca2+-uniporter also reduced normoxic AMPAR currents by 40%, and including BAPTA in the recording abolished the anoxia or agonist-mediated decrease. Therefore, the mKATP channel is involved in the anoxia-mediated down-regulation of AMPAR activity and is a common mechanism to reduce glutamatergic excitability.

Mammalian neurons rapidly undergo excitotoxic cell death during anoxia, while neurons from the anoxia-tolerant painted turtle can survive without oxygen for hours without apparent damage. An anoxia-mediated decrease in AMPA receptor currents are an important part of the turtle’s natural defence however the mechanism underlying it is unknown. Here I investigate a mechanism that involves activation of a mitochondrial KATP channel that subsequently signals a decrease in AMPAR currents. Whole-cell AMPAR currents were stable during normoxia, but anoxia or pharmacological activation of mKATP channels resulted in a 50% decrease in AMPAR currents. Conversely, mKATP antagonists blocked the anoxia-mediated decrease. Mitochondrial KCa channel modulators responded similarly. Blocking the Ca2+-uniporter also reduced normoxic AMPAR currents by 40%, and including BAPTA in the recording abolished the anoxia or agonist-mediated decrease. Therefore, the mKATP channel is involved in the anoxia-mediated down-regulation of AMPAR activity and is a common mechanism to reduce glutamatergic excitability.

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.

Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.

Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.