Many vertebrates are challenged by either chronic or acute episodes of low oxygen availability in their natural environments. Brain function is especially vulnerable to the effects of hypoxia and can be irreversibly impaired by even brief periods of low oxygen supply. This review describes recent research on physiological mechanisms that have evolved in certain vertebrate species to cope with brain hypoxia. Four model systems are considered: freshwater turtles that can survive for months trapped in frozen-over lakes, arctic ground squirrels that respire at extremely low rates during winter hibernation, seals and whales that undertake breath-hold dives lasting minutes to hours, and naked mole-rats that live in crowded burrows completely underground for their entire lives. These species exhibit remarkable specializations of brain physiology that adapt them for acute or chronic episodes of hypoxia. These specializations may be reactive in nature, involving modifications to the catastrophic sequelae of oxygen deprivation that occur in non-tolerant species, or preparatory in nature, preventing the activation of those sequelae altogether. Better understanding of the mechanisms used by these hypoxiatolerant vertebrates will increase appreciation of how nervous systems are adapted for life in specific ecological niches as well as inform advances in therapy for neurological conditions such as stroke and epilepsy.KEY WORDS: Arctic ground squirrel, Cetacean, Hypoxia, Naked mole-rat, Seal, Turtle IntroductionEnvironmental conditions vary enormously for vertebrates, both with respect to the extreme conditions tolerated by a given species at different times and with respect to average living conditions tolerated by different species. Temperature is perhaps the most obvious example: from the poles to the equator, average ambient temperatures vary widely and have been accompanied by physiological adaptations appropriate to resident species; seasonal variations in temperature and resource variability can induce dramatic changes in physiological and/or behavioral patterns including migration and hibernation. Oxygen levels also vary widely, with animals adapted to sea level, high-altitude, underground and aquatic habitats. Oxygen levels can also change dramatically on a shorter-term basis, as can occur in tidal pools or in breath-hold divers. Approximately 20% of the oxygen consumed by the human body is used by the brain. The greater part of this oxygen is used to produce the ATP required to maintain the membrane potentials necessary for electrical signaling with synaptic and action potentials (Harris et al., 2012). In many vertebrates, including adult humans, interruption of the oxygen supply to the brain for more than a few minutes leads to irreversible neurological damage, including neuronal death. Without oxidative phosphorylation, ATP-dependent neuronal processes including ion transport and neurotransmitter reuptake decline sharply. Without pumping, ion gradients fail and neurons depolarize, releasing excessive levels...

The turtle Trachemys scripta is one of a limited group of vertebrates that can withstand hours to days without oxygen. One facet of anoxic survival is the turtle's ability to maintain basal extracellular glutamate levels, whereas in most vertebrates, anoxia triggers massive excitotoxic glutamate release. We investigated glutamate release and reuptake in the anoxic turtle and the effects of adenosine and ATP-sensitive potassium (K(ATP)) channels on glutamate homeostasis. Striatal extracellular glutamate was measured in anesthetized T. scripta by microdialysis in normoxia and over 2-h anoxia. Glutamate release is decreased by 44% in the early anoxic turtle; this anoxia-induced decrease in glutamate release was prevented when K(ATP) channels and adenosine receptors were blocked simultaneously but not when either mechanism was blocked individually. We hypothesize that the continued release and reuptake of glutamate during anoxia help maintain neuronal tone and aid in the recovery of a functional neuronal network after long periods of anoxia, whereas activation of adenosine and/or K(ATP) conserves energy by reducing glutamate release and lowering transport costs.

Two naturally occurring strains of adult Drosophila melanogaster Meigen were used in this study: a rover strain homozygous for the for R allele (high PKG activity), and a sitter strain homozygous for for s (low PKG activity). These strains are isogenized natural polymorphisms of the foraging gene, located on the second chromosome (Sokolowski, 1980;Fitzpatrick et al., 2007). Additionally, the sitter mutant strain for s2, which was previously Accepted 12 April 2010 SUMMARY In this study we identify a cGMP-dependent protein kinase (PKG) cascade as a biochemical pathway critical for controlling lowoxygen tolerance in the adult fruit fly, Drosophila melanogaster. Even though adult Drosophila can survive in 0% oxygen (anoxia) environments for hours, air with less than 2% oxygen rapidly induces locomotory failure resulting in an anoxic coma. We use natural genetic variation and an induced mutation in the foraging (for) gene, which encodes a Drosophila PKG, to demonstrate that the onset of anoxic coma is correlated with PKG activity. Flies that have lower PKG activity demonstrate a significant increase in time to the onset of anoxic coma. Further, in vivo pharmacological manipulations reveal that reducing either PKG or protein phosphatase 2A (PP2A) activity increases tolerance of behavior to acute hypoxic conditions. Alternatively, PKG activation and phosphodiesterase (PDE5/6) inhibition significantly reduce the time to the onset of anoxic coma. By manipulating these targets in paired combinations, we characterized a specific PKG cascade, with upstream and downstream components. Further, using genetic variants of PKG expression/activity subjected to chronic anoxia over 6h, ~50% of animals with higher PKG activity survive, while only ~25% of those with lower PKG activity survive after a 24h recovery. Therefore, in this report we describe the PKG pathway and the differential protection of function vs survival in a critically low oxygen environment.

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H 2 O 2 increase after anoxia/ reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro, while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H 2 O 2 ), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.

The freshwater turtle Trachemys scripta is among the most anoxia-tolerant of vertebrates, a true facultative anaerobe able to survive without oxygen for days at room temperature to weeks or months during winter hibernation. Our good friend and colleague Peter Lutz devoted nearly 25 years to the study of the physiology of anoxia tolerance in these and other model organisms, promoting not just the basic science but also the idea that understanding the physiology and molecular mechanisms behind anoxia tolerance provides insights into critical survival pathways that may be applicable to the hypoxic/ischemic mammalian brain. Work by Peter and his colleagues focused on the factors which enable the turtle to enter a deep hypometabolic state, including decreases in ion flux ("channel arrest"), increases in inhibitory neuromodulators like adenosine and GABA, and the maintenance of low extracellular levels of excitatory compounds such as dopamine and glutamate. Our attention has recently turned to molecular mechanisms of anoxia tolerance, including the upregulation of such protective factors as heat shock proteins (Hsp72, Hsc73), the reversible downregulation of voltage gated potassium channels, and the modulation of MAP kinase pathways. In this review we discuss three phases of anoxia tolerance, including the initial metabolic downregulation over the first several hours, the long-term maintenance of neuronal function over days to weeks of anoxia, and finally recovery upon reoxygenation, with necessary defenses against reactive oxygen stress.