Environmental remodelling of GABAergic and glutamatergic neurotransmission: Rise of the anoxia-tolerant turtle brain

Climate cooling over the past one hundred thousand years has resulted in seasonal ice cover at northern and southern latitudes that has selected for hypoxia and anoxia tolerance in some species, such as freshwater turtles. At the northern reaches of their range, North American freshwater turtles spend 4 months or more buried in the mud bottom of ice covered lakes and ponds. From a comparative perspective this gives us the opportunity to understand how an extremely oxygen-sensitive organ, such as the vertebrate brain, can function without oxygen for long periods. Brain function is based on complex excitatory (on) and inhibitory (off) circuits involving the major neurotransmitters glutamate and, γ-aminobutyric acid (GABA) respectively. When a mammalian brain becomes anoxic, glutamate levels rise within minutes resulting in excitotoxic cell death which does not occur in anoxic turtle brain. The response in turtle brain has been remodelled – GABA levels rise rapidly resulting in large inhibitory GABA receptor currents and inhibition of glutamate receptor function that together depress neuronal activity.     

Antioxidant status of anoxia-tolerant and -intolerant plant species under anoxia and reaeration

The redox potential of the cell, as well as the antioxidant status of the tissue, are considered to be important regulatory constituents in an adaptive response in plants. Here the involvement of active antioxidants ascorbic acid (AA), reduced glutathione (GSH) and α - and β -tocopherols in reactive oxygen species scavenging, and the effect of anoxic stress on their reduction state were studied in 4 anoxia-tolerant and -intolerant plant species: Iris germanica L., Iris pseudacorus L., wheat ( Triticum aestivum L. cv. Leningradka) and rice ( Oryza sativa L. cv. VNIIR). The initial antioxidant content (both AA and GSH) was higher in the rhizomes of the more anoxia-tolerant Iris spp., as compared with that of the roots of the cereals. The predominant form of ascorbate was dehydroascorbic acid (DHA) in the cereals and AA in the Iris spp. Imposition of anoxia with subsequent reoxygenation resulted in an overall depletion of the reduced forms of antioxidants. No concurrent increase in oxidised forms (DHA and conjugated glutathione) was observed in anoxic samples. α -tocopherol content in Iris spp. was in the range 1–2 μg g⁻¹ fresh weight, while β -tocopherol content was higher in the anoxia-intolerant I. germanica (7.2 μg g⁻¹ fresh weight) as compared with the tolerant I. pseudacorus (1.5 μg g⁻¹ fresh weight). In I. pseudacorus , a significant decrease in α - and β -tocopherol levels was observed only after long-term (45 days) anoxia. The results suggested exclusion of AA and GSH from the redox cycling under prolonged anoxia, and a concomitant decrease in the redox state, as well as an anoxia-induced depletion of α - and β -tocopherols.     

Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxia-tolerant turtle Trachemys scripta

Neuroglobin (Ngb) is an oxygen binding heme protein found in nervous tissue with a yet unclear physiological and protective role in the hypoxia-sensitive mammalian brain. Here we utilized in vivo and in vitro studies to examine the role of Ngb in anoxic and post-anoxic neuronal survival in the freshwater turtle. We employed semiquantitative RT-PCR and western blotting to analyze Ngb mRNA and protein levels in turtle brain and neuronally enriched cultures. Ngb expression is strongly up-regulated by hypoxia and post-anoxia reoxygenation but increases only modestly in anoxia. The potential neuroprotective role of Ngb in this species was analyzed by knocking down Ngb using specific small interfering RNA. Ngb knockdown in neuronally enriched cell cultures resulted in significant increases in H₂O₂ release compared to controls but no change in cell death. Cell survival may be linked to activation of other protective responses such as the extracellular regulated kinase transduction pathway, as phosphorylated extracellular regulated kinase levels in anoxia were significantly higher in Ngb knockdown cultures compared to controls. The greater expression of Ngb when reactive oxygen species are likely to be high, and the increased susceptibility of neurons to H₂O₂ release and external oxidative stress in knockdown cultures, suggests a role for Ngb in reducing reactive oxygen species production or in detoxification, though it does not appear to be of primary importance in the anoxia tolerant turtle in the presence of compensatory survival mechanisms.     

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.     

Effect of anoxia and pharmacological anoxia on whole-cell NMDA receptor currents in cortical neurons from the western painted turtle

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca(2+) entry via N-methyl-D-aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close-to-normal [Ca(2+)](i) during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca(2+) injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole-cell NMDAR currents respond similarly to true anoxia with N(2) and to NaCN-induced anoxia. Whole-cell NMDAR currents were measured during a control N(2)-induced anoxic transition and a control NaCN-induced transition. During anoxia with N(2) normalized, NMDAR currents decreased to 35.3%+/-10.8% of control values. Two different NMDAR current responses were observed during NaCN-induced anoxia: one resulted in a 172%+/-51% increase in NMDAR currents, and the other was a decrease to 48%+/-14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole-cell NMDAR currents respond differently to NaCN-induced anoxia than to the more physiologically relevant anoxia with N(2).     

Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta

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 H₂O₂ 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 (H₂O₂), 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.     

Antioxidant systems and anoxia tolerance in a freshwater turtle Trachemys scripta elegans

In the search for MBP phosphorylating activities in Dictyostelium discoideum, we have found a proteolysis-activated protein kinase. This activity which is distributed between the soluble and the particulate fractions of the cell, uses MBP and histone as substrate and has a molecular mass of 140 kDa as detected in an in situ' assay.This protein kinase has several features shared by the protein kinase C family, such as substrate specificity and sensitivity to proteolysis, but its molecular mass is much larger than that described for the known protein kinase C isoforms.To better characterize this activity we have studied its sensitivity to several protein kinase C inhibitors and activators. This protein kinase is activated neither by phorbol ester nor by phosphatidylserine or Ca²⁺. The activity is inhibited by staurosporine and PKC pseudosubstrate, but is not affected by the specific protein kinase C inhibitor bisindolylmaleimide.These data lead us to propose that proteolytically activated Dictyostelium protein kinase belongs to the recently described protein kinase C-related family.     

Beyond apoptosis: nonapoptotic cell death in physiology and disease.

Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways, including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.     

Stress response in Drosophila subobscura. II. Puff activity during anoxia and recovery from anoxia

When individuals of Drosophila subobscura at 0 hr prepupa are submitted to anoxia (4 hr and 24 hr, respectively), their puffing pattern is very similar to that shown by individuals at the moment of development in which treatment began. The same expression of genes (the same puffing pattern and the same protein pattern) is induced in this species by recovery from anoxia as well as by heat shock treatment at 31 degrees C.