Maintained mitochondrial integrity without oxygen in the anoxia-tolerant crucian carp.

Very few vertebrates survive without oxygen (anoxia) for more than a few minutes. Crucian carp (Carassius carassius) are one example, surviving months of anoxia at low temperatures, and we hypothesised that they maintain mitochondrial membrane potential and function. Isolated crucian carp cardiomyocytes indeed maintained mitochondrial membrane potential after blocking complex IV of the electron transport system with cyanide, while those of anoxia-intolerant trout depolarised. When complexes I-III were inhibited, crucian carp mitochondria depolarised, indicating that these complexes need to function during anoxia. Mitochondrial membrane potential depended on reversal of ATP synthase in chemical anoxia, as blocking with cyanide combined with oligomycin to inhibit ATP synthase led to depolarisation. ATP synthase activity was reduced in the heart after 1 week of anoxia in crucian carp, together with a downregulation of ATP synthase subunit gene expression. However, the morphology of cardiac mitochondria was not affected by 1 week of anoxia, even with a large increase in mitofusin 2 mRNA expression. Cardiac citrate synthase activity was not affected by anoxia, while cytochrome c oxidase activity was increased. We show how mitochondria respond to anoxia. A mechanistic understanding of how mitochondrial function can be maintained in anoxia may provide new perspectives to reduce mitochondrial damage in anoxia-sensitive organisms.

Responses of neurogenesis and neuroplasticity related genes to elevated CO2 levels in the brain of three teleost species.

The continuous increase of anthropogenic CO2 in the atmosphere resulting in ocean acidification has been reported to affect brain function in some fishes. During adulthood, cell proliferation is fundamental for fish brain growth and for it to adapt in response to external stimuli, such as environmental changes. Here we report the first expression study of genes regulating neurogenesis and neuroplasticity in brains of three-spined stickleback (Gasterosteus aculeatus), cinnamon anemonefish (Amphiprion melanopus) and spiny damselfish (Acanthochromis polyacanthus) exposed to elevated CO2 The mRNA expression levels of the neurogenic differentiation factor (NeuroD) and doublecortin (DCX) were upregulated in three-spined stickleback exposed to high-CO2 compared with controls, while no changes were detected in the other species. The mRNA expression levels of the proliferating cell nuclear antigen (PCNA) and the brain-derived neurotrophic factor (BDNF) remained unaffected in the high-CO2 exposed groups compared to the control in all three species. These results indicate a species-specific regulation of genes involved in neurogenesis in response to elevated ambient CO2 levels. The higher expression of NeuroD and DCX mRNA transcripts in the brain of high-CO2-exposed three-spined stickleback, together with the lack of effects on mRNA levels in cinnamon anemonefish and spiny damselfish, indicate differences in coping mechanisms among fish in response to the predicted-future CO2 level.

Expression of genes involved in GABAergic neurotransmission in anoxic crucian carp brain (Carassius carassius).

The crucian carp, Carassius carassius, survives days to months without oxygen, depending on temperature. In the anoxic crucian carp brain, increased GABAergic inhibition, mediated by increased extracellular levels of GABA, has been shown to suppress electric activity and ATP consumption. To investigate an involvement of gene expression in this response, we utilized real-time RT-PCR to test the effect of 1 and 7 days anoxia (8 degrees C) on the expression of 22 genes, including nine GABA(A) receptor subunits (alpha(1-6), beta(2), delta, and gamma(2)), three GABA(B) receptor subunits (G(B)1a-1b and G(B)2), three enzymes involved in GABA metabolism (GAD65 and GAD67, GABAT), four GABA transporters (GAT1, 2a-b and 3), two GABA(A) receptor-associated proteins (GABARAP 1 and 2), and the K(+)/Cl(-) cotransporter KCC2. While the expression of GABA(A) receptor subunits was dominated by alpha(4)-, alpha(6)-, and delta-subunits, all of which are located to extrasynaptic sites in mammalian brains and respond to elevations in extracellular levels of GABA by showing tonic activity patterns, the expression of GABA transporters was dominated by GAT2 (a and b) and GAT3, which also show extrasynaptic location in mammals. These expression patterns differ from those observed in mammals and may be a prerequisite for GABAergic inhibition of anoxic metabolic rate in crucian carp. Furthermore, while the expression of the majority of the genes was largely unaltered by anoxia, the expression of GAT2 and GAT3 decreased to 20%. This suggests impairment of GABA transport, which could be a mechanism behind the accumulation of extracellular GABA and the increased GABAergic inhibition.

Erratum: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2.

[This corrects the article DOI: 10.1093/conphys/cow068.][This corrects the article DOI: 10.1093/conphys/cow068.].

Extreme anoxia tolerance in crucian carp and goldfish through neofunctionalization of duplicated genes creating a new ethanol-producing pyruvate decarboxylase pathway.

Without oxygen, most vertebrates die within minutes as they cannot meet cellular energy demands with anaerobic metabolism. However, fish of the genus Carassius (crucian carp and goldfish) have evolved a specialized metabolic system that allows them to survive prolonged periods without oxygen by producing ethanol as their metabolic end-product. Here we show that this has been made possible by the evolution of a pyruvate decarboxylase, analogous to that in brewer's yeast and the first described in vertebrates, in addition to a specialized alcohol dehydrogenase. Whole-genome duplication events have provided additional gene copies of the pyruvate dehydrogenase multienzyme complex that have evolved into a pyruvate decarboxylase, while other copies retained the essential function of the parent enzymes. We reveal the key molecular substitution in duplicated pyruvate dehydrogenase genes that underpins one of the most extreme hypoxic survival strategies among vertebrates and that is highly deleterious in humans.

Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2.

Change in the activity of the main inhibitory receptor, GABAA, has been suggested to be a general mechanism behind the behavioural alterations reported in ocean acidification studies on fish. It has been proposed that regulatory acid-base mechanisms in response to high CO2 alter the neuronal Cl- and HCO3- gradients that are important for GABAA receptor function. Here, we report a comprehensive analysis of gene expression of GABAA receptor subunits and of genes involved in GABAergic transmission in the brain of fish exposed to near-future CO2. Altogether, 56 mRNA transcripts were quantified in brains of three-spined stickleback (Gasterosteus aculeatus) kept in control pCO2 (333 ± 30 µatm CO2) or at high pCO2 levels (991 ± 57 µatm) for 43 days. The gene expression analysis included GABAA receptor subunits (α1-6, ß1-3, γ1-3, δ, π and ρ1-3), enzymes and transporters involved in GABA metabolism (GAD1-2, GABAT and GAT1-3), GABAA receptor-associated proteins (GABARAP and GABARAPL), ion cotransporters (KCC1-4, NKCC1, ClC21-3, AE3 and NDAE) and carbonic anhydrase (CAII). Exposure to high CO2 had only minor effects on the expression of genes involved in GABAergic neurotransmission. There were significant increases in the mRNA levels of α family subunits of the GABAA receptor, with a more pronounced expression of α12, α3, α4 and α6b. No changes were detected in the expression of other GABAA subunits or in genes related to receptor turnover, GABA metabolism or ion transport. Although the minor changes seen for mRNA levels might reflect compensatory mechanisms in the high-CO2 conditions, these were apparently insufficient to restore normal neural function, because the behavioural changes persisted within the time frame studied.

Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization.

The mRNA expression of heat-shock protein 90 (HSP90) and heat-shock cognate 70 (HSC70) was examined in cardiac chambers and telencephalon of warm- (21°C) and cold-acclimated (5°C) turtles (Trachemys scripta) exposed to normoxia, prolonged anoxia or anoxia followed by reoxygenation. Additionally, the suitability of total RNA as well as mRNA from ß-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin A (PPIA) for normalizing gene expression data was assessed, as compared to the use of an external RNA control. Measurements of HSP90 and HSC70 mRNA expression revealed that anoxia and reoxygenation have tissue- and gene-specific effects. By and large, the alterations support previous investigations on HSP protein abundance in the anoxic turtle heart and brain, as well as the hypothesized roles of HSP90 and HSC70 during stress and non-stress conditions. However, more prominent was a substantially increased HSP90 and HSC70 mRNA expression in the cardiac chambers with cold acclimation. The finding provides support for the notion that cold temperature induces a number of adaptations in tissues of anoxia-tolerant vertebrates that precondition them for winter anoxia. ß-actin, GAPDH and PPIA mRNA expression and total RNA also varied with oxygenation state and acclimation temperature in a tissue- and gene-specific manner, as well as among tissue types, thus disqualifying them as suitable for real-time RT-PCR normalization. Thus, the present data highlights the advantages of normalizing real-time RT-PCR data to an external RNA control, an approach that also allows inter-tissue and potentially inter-species comparisons of target gene expression.