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.

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.