Alfaxalone is an effective anesthetic for the electrophysiological study of anoxia-tolerance mechanisms in western painted turtle pyramidal neurons.

Anoxia in the mammalian brain leads to hyper-excitability and cell death; however, this cascade of events does not occur in the anoxia-tolerant brain of the western painted turtle, Chrysemys picta belli. The painted turtle has become an important anoxia-tolerant model to study brain, heart, and liver function in the absence of oxygen, but being anoxia-tolerant likely means that decapitation alone is not a suitable method of euthanasia. Many anesthetics have long-term effects on ion channels and are not appropriate for same day experimentation. Using whole-cell electrophysiological techniques, we examine the effects of the anesthetic, Alfaxalone, on pyramidal cell action potential amplitude, threshold, rise and decay time, width, frequency, whole cell conductance, and evoked GABAA receptors currents to determine if any of these characteristics are altered with the use of Alfaxalone for animal sedation. We find that Alfaxalone has no long-term impact on action potential parameters or whole-cell conductance. When acutely applied to naïve tissue, Alfaxalone did lengthen GABAA receptor current decay rates by 1.5-fold. Following whole-animal sedation with Alfaxalone, evoked whole cell GABAA receptor current decay rates displayed an increasing trend with 1 and 2 hours after brain sheet preparation, but showed no significant change after a 3-hour washout period. Therefore, we conclude that Alfaxalone is a suitable anesthetic for same day use in electrophysiological studies in western painted turtle brain tissue.

Evolution of a novel regulatory mechanism of hypoxia inducible factor in hypoxia-tolerant electric fishes.

Hypoxia is a significant source of metabolic stress that activates many cellular pathways involved in cellular differentiation, proliferation, and cell death. Hypoxia is also a major component in many human diseases and a known driver of many cancers. Despite the challenges posed by hypoxia, there are animals that display impressive capacity to withstand lethal levels of hypoxia for prolonged periods of time and thus offer a gateway to a more comprehensive understanding of the hypoxic response in vertebrates. The weakly electric fish genus Brachyhypopomus inhabits some of the most challenging aquatic ecosystems in the world, with some species experiencing seasonal anoxia, thus providing a unique system to study the cellular and molecular mechanisms of hypoxia tolerance. In this study, we use closely related species of Brachyhypopomus that display a range of hypoxia tolerances to probe for the underlying molecular mechanisms via hypoxia inducible factors (HIFs)-transcription factors known to coordinate the cellular response to hypoxia in vertebrates. We find that HIF1⍺ from hypoxia tolerant Brachyhypopomus species displays higher transactivation in response to hypoxia than that of intolerant species, when overexpressed in live cells. Moreover, we identified two SUMO-interacting motifs near the oxygen-dependent degradation and transactivation domains of the HIF1⍺ protein that appear to boost transactivation of HIF1, regardless of the genetic background. Together with computational analyses of selection, this shows that evolution of HIF1⍺ are likely to underlie adaptations to hypoxia tolerance in Brachyhypopomus electric fishes, with changes in two SUMO-interacting motifs facilitating the mechanism of this tolerance.

Skeletal muscle metabolism and contraction performance regulation by teneurin C-terminal-associated peptide-1.

Skeletal muscle regulation is responsible for voluntary muscular movement in vertebrates. The genes of two essential proteins, teneurins and latrophilins (LPHN), evolving in ancestors of multicellular animals form a ligand-receptor pair, and are now shown to be required for skeletal muscle function. Teneurins possess a bioactive peptide, termed the teneurin C-terminal associated peptide (TCAP) that interacts with the LPHNs to regulate skeletal muscle contractility strength and fatigue by an insulin-independent glucose importation mechanism in rats. CRISPR-based knockouts and siRNA-associated knockdowns of LPHN-1 and-3 in the C2C12 mouse skeletal cell line shows that TCAP stimulates an LPHN-dependent cytosolic Ca2+ signal transduction cascade to increase energy metabolism and enhance skeletal muscle function via increases in type-1 oxidative fiber formation and reduce the fatigue response. Thus, the teneurin/TCAP-LPHN system is presented as a novel mechanism that regulates the energy requirements and performance of skeletal muscle.

Exposure to low temperature prepares the turtle brain to withstand anoxic environments during overwintering.

In most vertebrates, anoxia drastically reduces the production of the essential adenosine triphosphate (ATP) to power its many necessary functions, and, consequently, cell death occurs within minutes. However, some vertebrates, such as the painted turtle (Chrysemys picta bellii), have evolved the ability to survive months without oxygen by simultaneously decreasing ATP supply and demand, surviving the anoxic period without any apparent cellular damage. The impact of anoxia on the metabolic function of painted turtles has received a lot of attention. However, the impact of low temperature has received less attention and the interactive effect of anoxia and temperature even less. In the present study, we investigated the interactive impacts of reduced temperature and severe hypoxia on the electrophysiological properties of pyramidal neurons in painted turtle cerebral cortex. Our results show that an acute reduction in temperature from 20 to 5°C decreases membrane potential, action potential width and amplitude, and whole-cell conductance. Importantly, acute exposure to 5°C considerably slows membrane repolarization by voltage-gated K+ channels. Exposing pyramidal cells to severe hypoxia in addition to an acute temperature change slightly depolarized membrane potential but did not alter action potential amplitude or width and whole-cell conductance. These results suggest that acclimation to low temperatures, preceding severe environmental hypoxia, induces cellular responses in pyramidal neurons that facilitate survival under low oxygen concentrations. In particular, our results show that temperature acclimation invokes a change in voltage-gated K+ channel kinetics that overcomes the acute inhibition of the channel.

Taurine activates glycine and GABAA receptor currents in anoxia-tolerant painted turtle pyramidal neurons.

Unlike anoxia-intolerant mammals, painted turtles can survive extended periods without oxygen. This is partly accomplished by an anoxia-mediated increase in gamma-aminobutyric acid (GABA) release, which activates GABA receptors and mediates spike arrest in turtle neurons via shunting inhibition. Extracellular taurine levels also increase during anoxia; why this occurs is unknown but it is speculated that glycine and/or GABAA/B receptors are involved. Given the general importance of inhibitory neurotransmission in the anoxia-tolerant painted turtle brain, we investigated the function of taurine as an inhibitory neuromodulator in turtle pyramidal neurons. Using whole-cell patch-clamp electrophysiological methods to record from neurons within a cortical brain sheet, we found that taurine depolarized membrane potential by ∼8 mV, increased whole-cell conductance ∼2-fold, and induced an inward current that possessed characteristics similar to GABA- and glycine-evoked currents. These effects were mitigated following glycine receptor antagonism with strychnine and GABAA receptor antagonism with gabazine, bicuculine or picrotoxin, but were unchanged following GABAB or glutamatergic receptor inhibition. These data indicate that a high concentration of taurine in vitro mediates its effects through both glycine and GABAA receptors, and suggests that taurine, in addition to GABA, inhibits neuronal activity during anoxia in the turtle cortex.

The hypoxia-tolerant vertebrate brain: Arresting synaptic activity.

The ion channel arrest hypothesis has been the foundation of three decades of research into the underlying mechanisms of hypoxia/anoxia tolerance in several key species, including: painted turtles, goldfish, crucian carp, naked mole rats, and arctic and ground squirrels. The hypothesis originally stated that hypoxia/anoxia tolerant species ought to have fewer ion channels per area membrane and/or mechanisms to regulate the conductance of ion channels. Today we can add to this and include mechanisms to remove channels from membranes and the expression of low conductance isoforms. Furthermore, possible oxygen sensing mechanisms in brain include a link to mitochondrial function, changes in the concentration of intracellular Ca2+ and reactive oxygen species, and activation of protein kinase C and a phosphatase. Importantly ion channel arrest leads to a decrease in metabolic rate that is fundamental to survival without oxygen and in brain is reflected in decreased action potential frequency or spike arrest. This results not only from a decrease in excitatory glutamatergic receptor currents but also by an increase in inhibitory GABAergic receptor currents. The surprising finding that ionic conductance through some ion channels increases is novel and contrary to the ion channel arrest hypothesis. The major insight that this offers is that key regulatory events are occurring at the level of the synapse and we therefore propose the "synaptic arrest hypothesis".

Stellate and pyramidal neurons in goldfish telencephalon respond differently to anoxia and GABA receptor inhibition.

With oxygen deprivation, the mammalian brain undergoes hyper-activity and neuronal death while this does not occur in the anoxia-tolerant goldfish (Carassius auratus). Anoxic survival of the goldfish may rely on neuromodulatory mechanisms to suppress neuronal hyper-excitability. As γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, we decided to investigate its potential role in suppressing the electrical activity of goldfish telencephalic neurons. Utilizing whole-cell patch-clamp recording, we recorded the electrical activities of both excitatory (pyramidal) and inhibitory (stellate) neurons. With anoxia, membrane potential (Vm) depolarized in both cell types from -72.2 mV to -57.7 mV and from -64.5 mV to -46.8 mV in pyramidal and stellate neurons, respectively. While pyramidal cells remained mostly quiescent, action potential frequency (APf) of the stellate neurons increased 68-fold. Furthermore, the GABAA receptor reversal potential (E-GABA) was determined using the gramicidin perforated-patch-clamp method and found to be depolarizing in pyramidal (-53.8 mV) and stellate neurons (-42.1 mV). Although GABA was depolarizing, pyramidal neurons remained quiescent as EGABA was below the action potential threshold (-36 mV pyramidal and -38 mV stellate neurons). Inhibition of GABAA receptors with gabazine reversed the anoxia-mediated response. While GABAB receptor inhibition alone did not affect the anoxic response, co-antagonism of GABAA and GABAB receptors (gabazine and CGP-55848) led to the generation of seizure-like activities in both neuron types. We conclude that with anoxia, Vm depolarizes towards EGABA which increases APf in stellate neurons and decreases APf in pyramidal neurons, and that GABA plays an important role in the anoxia tolerance of goldfish brain.

Assessment of anoxia tolerance and photoperiod dependence of GABAergic polarity in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is reported to be anoxia-tolerant and if the tolerance mechanism is similar to that of the anoxia-tolerant painted turtle, GABA should play an important role. A potentially confounding factor investigating the role of GABA in anoxia tolerance are reports that GABA has both inhibitory and excitatory effects within L. stagnalis central ganglion. We therefore set out to determine if seasonality or photoperiod has an impact on: 1) the anoxia-tolerance of the intact pond snail, and 2) the response of isolated neuroganglia cluster F neurons to exogenous GABA application. L. stagnalis maintained on a natural summer light cycle were unable to survive any period of anoxic exposure, while those maintained on a natural winter light cycle survived a maximum of 4h. Using intracellular sharp electrode recordings from pedal ganglia cluster F neurons we show that there is a photoperiod dependent shift in the response to GABA. Snails exposed to a 16h:8h light:dark cycle in an environmental chamber (induced summer phenotype) exhibited hyperpolarizing inhibitory responses and those exposed to a 8h:16h light:dark cycle (induced winter phenotype) exhibited depolarizing excitatory responses to GABA application. Using gramicidin-perforated patch recordings we also found a photoperiod dependent shift in the reversal potential for GABA. We conclude that the opposing responses of L. stagnalis central neurons to GABA results from a shift in intracellular chloride concentration that is photoperiod dependent and is likely mediated through the relative efficacy of cation chloride co-transporters. Although the physiological ramifications of the photoperiod dependent shift are unknown this work potentially has important implications for the impact of artificial light pollution on animal health.

Sensing and surviving hypoxia in vertebrates.

Surviving hypoxia is one of the most critical challenges faced by vertebrates. Most species have adapted to changing levels of oxygen in their environment with specialized organs that sense hypoxia, while only few have been uniquely adapted to survive prolonged periods of anoxia. The goal of this review is to present the most recent research on oxygen sensing, adaptation to hypoxia, and mechanisms of anoxia tolerance in nonmammalian vertebrates. We discuss the respiratory structures in fish, including the skin, gills, and air-breathing organs, and recent evidence for chemosensory neuroepithelial cells (NECs) in these tissues that initiate reflex responses to hypoxia. The use of the zebrafish as a genetic and developmental model has allowed observation of the ontogenesis of respiratory and chemosensory systems, demonstration of a putative intracellular O2 sensor in chemoreceptors that may initiate transduction of the hypoxia signal, and investigation into the effects of extreme hypoxia on cardiorespiratory development. Other organisms, such as goldfish and freshwater turtles, display a high degree of anoxia tolerance, and these models are revealing important adaptations at the cellular level, such as the regulation of glutamatergic and GABAergic neurotransmission in defense of homeostasis in central neurons.

Transcriptomic Responses of the Heart and Brain to Anoxia in the Western Painted Turtle.

Painted turtles are the most anoxia-tolerant tetrapods known, capable of surviving without oxygen for more than four months at 3°C and 30 hours at 20°C. To investigate the transcriptomic basis of this ability, we used RNA-seq to quantify mRNA expression in the painted turtle ventricle and telencephalon after 24 hours of anoxia at 19°C. Reads were obtained from 22,174 different genes, 13,236 of which were compared statistically between treatments for each tissue. Total tissue RNA contents decreased by 16% in telencephalon and 53% in ventricle. The telencephalon and ventricle showed ≥ 2x expression (increased expression) in 19 and 23 genes, respectively, while only four genes in ventricle showed ≤ 0.5x changes (decreased expression). When treatment effects were compared between anoxic and normoxic conditions in the two tissue types, 31 genes were increased (≥ 2x change) and 2 were decreased (≤ 0.5x change). Most of the effected genes were immediate early genes and transcription factors that regulate cellular growth and development; changes that would seem to promote transcriptional, translational, and metabolic arrest. No genes related to ion channels, synaptic transmission, cardiac contractility or excitation-contraction coupling changed. The generalized expression pattern in telencephalon and across tissues, but not in ventricle, correlated with the predicted metabolic cost of transcription, with the shortest genes and those with the fewest exons showing the largest increases in expression.

Decreases in mitochondrial reactive oxygen species initiate GABA(A) receptor-mediated electrical suppression in anoxia-tolerant turtle neurons.

Anoxia induces hyper-excitability and cell death in mammalian brain but in the anoxia-tolerant western painted turtle (Chrysemys picta bellii) neuronal electrical activity is suppressed (i.e. spike arrest), adenosine triphosphate (ATP) consumption is reduced, and cell death does not occur. Electrical suppression is primarily the result of enhanced γ-aminobutyric acid (GABA) transmission; however, the underlying mechanism responsible for initiating oxygen-sensitive GABAergic spike arrest is unknown. In turtle cortical pyramidal neurons there are three types of GABA(A) receptor-mediated currents: spontaneous inhibitory postsynaptic currents (IPSCs), giant IPSCs and tonic currents. The aim of this study was to assess the effects of reactive oxygen species (ROS) scavenging on these three currents since ROS levels naturally decrease with anoxia and may serve as a redox signal to initiate spike arrest. We found that anoxia, pharmacological ROS scavenging, or inhibition of mitochondrial ROS generation enhanced all three types of GABA currents, with tonic currents comprising ∼50% of the total current. Application of hydrogen peroxide inhibited all three GABA currents, demonstrating a reversible redox-sensitive signalling mechanism. We conclude that anoxia-mediated decreases in mitochondrial ROS production are sufficient to initiate a redox-sensitive inhibitory GABA signalling cascade that suppresses electrical activity when oxygen is limited. This unique strategy for reducing neuronal ATP consumption during anoxia represents a natural mechanism in which to explore therapies to protect mammalian brain from low-oxygen insults.

Proteomic changes in the brain of the western painted turtle (Chrysemys picta bellii) during exposure to anoxia.

During anoxia, overall protein synthesis is almost undetectable in the brain of the western painted turtle. The aim of this investigation was to address the question of whether there are alterations to specific proteins by comparing the normoxic and anoxic brain proteomes. Reductions in creatine kinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase reflected the reduced production of adenosine triphosphate (ATP) during anoxia while the reduction in transitional endoplasmic reticulum ATPase reflected the conservation of ATP or possibly a decrease in intracellular Ca(2+). In terms of neural protection programed cell death 6 interacting protein (PDCD6IP; a protein associated with apoptosis), dihydropyrimidinase-like protein, t-complex protein, and guanine nucleotide protein G(o) subunit alpha (Go alpha; proteins associated with neural degradation and impaired cognitive function) also declined. A decline in actin, gelsolin, and PDCD6IP, together with an increase in tubulin, also provided evidence for the induction of a neurological repair response. Although these proteomic alterations show some similarities with the crucian carp (another anoxia-tolerant species), there are species-specific responses, which supports the theory of no single strategy for anoxia tolerance. These findings also suggest the anoxic turtle brain could be an etiological model for investigating mammalian hypoxic damage and clinical neurological disorders.

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage.

BACKGROUND: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing. RESULTS: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented. CONCLUSIONS: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Oxygen sensitive synaptic neurotransmission in anoxia-tolerant turtle cerebrocortex.

Anoxia rapidly elicits hyper-excitability and cell death in mammal brain but this is not so in anoxia-tolerant turtle brain where spontaneous electrical activity is suppressed by anoxia (i.e. spike arrest; SA). In anoxic turtle brain extracellular GABA concentrations increase dramatically and impact GABAergic synaptic transmission in a way that results in SA. Here we briefly review what is known about the regulation of glutamatergic signalling during anoxia and investigate the possibility that in anoxic turtle cortical neurons GABA(A/B) receptors play an important role in neuroprotection. Both AMPA and NMDA receptor currents decrease by about 50% in anoxic turtle cerebrocortex and therefore exhibit channel arrest, whereas GABA-A receptor currents increase twofold and increase whole-cell conductance. The increased post synaptic GABA-A receptor current is contrary to the channel arrest hypothesis but it does serve an important function. The reversal potential of the GABA-A receptor (E(GABA)) is only slightly depolarized relative to the resting membrane potential of the neuron and not sufficient to elicit an action potential. Therefore, when GABA-A receptors are activated, membrane potential moves to E(GABA) and prevents further depolarization by glutamatergic inputs during anoxia by a process termed shunting inhibition. Furthermore we discuss the presynaptic role of GABA-B receptors and show that increased endogenous GABA release during anoxia mediates SA by activating both GABA-A and B receptors and that this represents a natural oxygen-sensitive adaptive mechanism to protect brain from anoxic injury.

The relationship between NMDA receptor function and the high ammonia tolerance of anoxia-tolerant goldfish.

Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-D-aspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96 h median lethal concentration (LC(50)) values of 199 µmol l(-1) and 4132 µmol l(-1) for NH(3) and total ammonia ([T(Amm)]=[NH(3)]+[NH(4)(+)]), respectively. These values were ~5-6 times greater than corresponding NH(3) and T(Amm) LC(50) values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH(4)Cl (3-5 mmol l(-1)) for 5 days, despite 6-fold increases in plasma [T] to ~1300 µmol l(-1) and 3-fold increases in brain [T(Amm)] to 6700 µmol l(-1). Muscle [T(Amm)] increased by almost 8-fold from ~900 µmol kg(-1) wet mass (WM) to greater than 7000 µmol kg(-1) WM by 48 h, and stabilized. Although urea excretion rates (J(Urea)) increased by 2-3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ~3000 µmol kg(-1) WM to 15,000 µmol kg(-1) WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5-8 mg kg(-1)) or ethanol (1-8 mg kg(-1)) increased trout survival time by 1.5-2.0-fold during exposure to 2 mmol l(-1) ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8-9.5 mmol l(-1) T(Amm)) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro exposure to 5 mmol l(-1) or 10 mmol l(-1) T(Amm) reversibly potentiated NMDA receptor currents. This observation suggested that goldfish neurons may not be completely resistant to ammonia-induced excitotoxicity. Subsequent western blot and densitometric analyses revealed that NMDA receptor NR1 subunit abundance was 40-60% lower in goldfish exposed to 3-5 mmol l(-1) T(Amm) for 5 days, which was followed by a restoration of NR1 subunit abundance after 3 days recovery in ammonia-free water. We conclude that the goldfish brain may be protected from excitotoxicity by downregulating the abundance of functional NMDA receptors during periods when it experiences increased internal ammonia.

Endogenous GABA(A) and GABA(B) receptor-mediated electrical suppression is critical to neuronal anoxia tolerance.

Anoxic insults cause hyperexcitability and cell death in mammalian neurons. Conversely, in anoxia-tolerant turtle brain, spontaneous electrical activity is suppressed by anoxia (i.e., spike arrest; SA) and cell death does not occur. The mechanism(s) of SA is unknown but likely involves GABAergic synaptic transmission, because GABA concentration increases dramatically in anoxic turtle brain. We investigated this possibility in turtle cortical neurons exposed to anoxia and/or GABA(A/B) receptor (GABAR) modulators. Anoxia increased endogenous slow phasic GABAergic activity, and both anoxia and GABA reversibly induced SA by increasing GABA(A)R-mediated postsynaptic activity and Cl(-) conductance, which eliminated the Cl(-) driving force by depolarizing membrane potential (∼8 mV) to GABA receptor reversal potential (∼-81 mV), and dampened excitatory potentials via shunting inhibition. In addition, both anoxia and GABA decreased excitatory postsynaptic activity, likely via GABA(B)R-mediated inhibition of presynaptic glutamate release. In combination, these mechanisms increased the stimulation required to elicit an action potential >20-fold, and excitatory activity decreased >70% despite membrane potential depolarization. In contrast, anoxic neurons cotreated with GABA(A+B)R antagonists underwent seizure-like events, deleterious Ca(2+) influx, and cell death, a phenotype consistent with excitotoxic cell death in anoxic mammalian brain. We conclude that increased endogenous GABA release during anoxia mediates SA by activating an inhibitory postsynaptic shunt and inhibiting presynaptic glutamate release. This represents a natural adaptive mechanism in which to explore strategies to protect mammalian brain from low-oxygen insults.

Matching cellular metabolic supply and demand in energy-stressed animals.

Certain environmental stressors can impair cellular ATP production to the point of harming or even killing an animal. Some exceptional animals employ strategies that maintain the balance between ATP production and consumption, allowing them to tolerate prolonged exposure to stressors such as hypoxia and anoxia. Anoxia- and hypoxia-tolerant animals reduce ATP consumption by ion-motive ATPases while concomitant reductions in passive ion flux reduce the demand for ion pumping and maintain transmembrane ion gradients. Reductions in gene transcription and protein turnover decrease ATP demand in hibernating and hypoxia-tolerant animals. Proton leak uncouples mitochondrial substrate oxidation from ATP synthesis and accounts for a considerable proportion of cellular energy demand, but there is little evidence that the proton permeability of inner mitochondrial membranes decreases in animals that tolerate energy stress. Indeed in some cases proton leak increases, possibly reducing reactive oxygen species production. Because substrate oxidation is important to the control of cellular metabolism, the downregulation of ATP supply pathways contributes significantly to metabolic suppression under energy stress. Mechanisms that coordinate the downregulation of both ATP supply and demand pathways include AMP kinase and ATP-sensitive ion channels. Strategies employed by animals tolerant to one energy stress often convey "cross-tolerance" to completely different stresses.

delta-Opioid receptor antagonism induces NMDA receptor-dependent excitotoxicity in anoxic turtle cortex.

delta-Opioid receptor (DOR) activation is neuroprotective against short-term anoxic insults in the mammalian brain. This protection may be conferred by inhibition of N-methyl-d-aspartate receptors (NMDARs), whose over-activation during anoxia otherwise leads to a deleterious accumulation of cytosolic calcium ([Ca(2+)](c)), severe membrane potential (E(m)) depolarization and excitotoxic cell death (ECD). Conversely, NMDAR activity is decreased by approximately 50% with anoxia in the cortex of the painted turtle, and large elevations in [Ca(2+)](c), severe E(m) depolarization and ECD are avoided. DORs are expressed in high quantity throughout the turtle brain relative to the mammalian brain; however, the role of DORs in anoxic NMDAR regulation has not been investigated in turtles. We examined the effect of DOR blockade with naltrindole (1-10 micromol l(-1)) on E(m), NMDAR activity and [Ca(2+)](c) homeostasis in turtle cortical neurons during normoxia and the transition to anoxia. Naltrindole potentiated normoxic NMDAR currents by 78+/-5% and increased [Ca(2+)](c) by 13+/-4%. Anoxic neurons treated with naltrindole were strongly depolarized, NMDAR currents were potentiated by 70+/-15%, and [Ca(2+)](c) increased 5-fold compared with anoxic controls. Following naltrindole washout, E(m) remained depolarized and [Ca(2+)](c) became further elevated in all neurons. The naltrindole-mediated depolarization and increased [Ca(2+)](c) were prevented by NMDAR antagonism or by perfusion of the G(i) protein agonist mastoparan-7, which also reversed the naltrindole-mediated potentiation of NMDAR currents. Together, these data suggest that DORs mediate NMDAR activity in a G(i)-dependent manner and prevent deleterious NMDAR-mediated [Ca(2+)](c) influx during anoxic insults in the turtle cortex.

Evidence of anoxia-induced channel arrest in the brain of the goldfish (Carassius auratus).

The common goldfish (Carassius auratus) is extremely anoxia tolerant and here we provide evidence that "channel arrest" in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca(2+)-channel, underwent a 40-50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L(-1)), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O(2)) provided indirect evidence that a reduction in Na(+)/K(+)-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na(+)/K(+)-ATPase activity of 30-40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na(+)/K(+)-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid "channel arrest" of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na(+)/K(+)-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.

Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability.

The ability of fishes, amphibians, and reptiles to survive extremes of oxygen availability derives from a core triad of adaptations: profound metabolic suppression, tolerance of ionic and pH disturbances, and mechanisms for avoiding free-radical injury during reoxygenation. For long-term anoxic survival, enhanced storage of glycogen in critical tissues is also necessary. The diversity of body morphologies and habitats and the utilization of dormancy have resulted in a broad array of adaptations to hypoxia in lower vertebrates. For example, the most anoxia-tolerant vertebrates, painted turtles and crucian carp, meet the challenge of variable oxygen in fundamentally different ways: Turtles undergo near-suspended animation, whereas carp remain active and responsive in the absence of oxygen. Although the mechanisms of survival in both of these cases include large stores of glycogen and drastically decreased metabolism, other mechanisms, such as regulation of ion channels in excitable membranes, are apparently divergent. Common themes in the regulatory adjustments to hypoxia involve control of metabolism and ion channel conductance by protein phosphorylation. Tolerance of decreased energy charge and accumulating anaerobic end products as well as enhanced antioxidant defenses and regenerative capacities are also key to hypoxia survival in lower vertebrates.