Remarkable neuronal hypoxia tolerance in the deep-diving adult hooded seal (Cystophora cristata)

doi:10.1016/j.neulet.2008.09.040

Neuroscience Letters

Volume 446, Issues 2–3, 3 December 2008, Pages 147-150

https://doi.org/10.1016/j.neulet.2008.09.040 Get rights and content

Abstract

Seals cope with regular exposure to diving hypoxia by storing oxygen in blood and skeletal muscles and by limiting the distribution of blood-borne oxygen to all but the most hypoxia vulnerable tissues (brain, heart), through dramatic cardiovascular adjustments. Still, arterial oxygen tension of freely diving seals regularly drops to levels that would be fatal to most non-diving mammals. Some cerebral protection is offered through diving-induced brain cooling and, possibly, enhanced oxygen delivery due to a particularly high brain capillary density. Here we test the hypothesis that seal neurons are in addition also intrinsically hypoxia tolerant. For this purpose we compared neuronal hypoxic responses in adult hooded seals and mice using intracellular recordings from the pyramidal layer of isolated visual cortex slices. Neurons from both species maintained normoxic membrane potentials of −60 to −70 mV, which in seals increased by only 13.4 ± 19.2 mV (n = 7) during the first 10 min of severe hypoxia (oxygen content of saline perfusate reduced from ∼75 to ∼5%), while the corresponding depolarization of mouse neurons was significantly larger (65.0 ± 44.9 mV; n = 14; p = 0.006). Mouse neurons moreover lost the ability to discharge after 5 ± 2 min in hypoxia, while seal neurons continued on average for 19 ± 10 min, in one case for a full hour. These results show that seal neocortical neurons exhibit a remarkable intrinsic hypoxia tolerance, which may partly explain why seals can dive for more than 1 h and stay alert without suffering from detrimental effects of hypoxia.

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Acknowledgements

The authors thank the crew of R/V Jan Mayen for assistance in the field and the Department of Medical Physiology, University of Tromsø, for lending us some of the instruments. This study was supported by grants from the Norwegian Research Council (no. 164791/V40) and the Roald Amundsen Centre for Arctic Research.

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While the brain of most mammals suffers from irreversible damage after only short periods of low oxygen levels (hypoxia), marine mammals are excellent breath-hold divers that have adapted to hypoxia. In addition to physiological adaptations, such as large oxygen storing capacity and strict oxygen economy during diving, the neurons of the deep-diving hooded seal (Cystophora cristata) have an intrinsic tolerance to hypoxia. We aim to understand the molecular basis of this neuronal hypoxia tolerance.
Previously, transcriptomics of the cortex of the hooded seal have revealed remarkably high expression levels of S100B and clusterin (apolipoprotein J) when compared to the ferret, a non-diving carnivore. Both genes have much-debated roles in hypoxia and oxidative stress. Here, we evaluated the effects of S100B and of two isoforms of clusterin (soluble and nucleus clusterin) on the survival, metabolic activity and the amount of reactive oxygen species (ROS) in HN33 neuronal mouse cells exposed to hypoxia and oxidative stress. S100B and soluble clusterin had neuroprotective effects, with reduced ROS-levels and retention of normoxic energy status of cells during both stress conditions. The protective effects of nucleus clusterin were restricted to hypoxia. S100B and clusterin showed purifying selection in marine and terrestrial mammals, indicating a functional conservation across species. Immunofluorescence revealed identical cellular distributions of S100B and clusterin in mice, ferrets and hooded seals, further supporting the functional conservation. Taken together, our data suggest that the neuroprotective effects of all three proteins are exclusively facilitated by their increased expression in the brain of the hooded seal.
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The brain of diving mammals is repeatedly exposed to low oxygen conditions (hypoxia) that would have caused severe damage to most terrestrial mammals. Some whales may dive for >2 h with their brain remaining active. Many of the physiological adaptations of whales to diving have been investigated, but little is known about the molecular mechanisms that enable their brain to survive sometimes prolonged periods of hypoxia. Here, we have used an RNA-Seq approach to compare the mRNA levels in the brains of whales with those of cattle, which serves as a terrestrial relative. We sequenced the transcriptomes of the brains from cattle (Bos taurus), killer whale (Orcinus orca), and long-finned pilot whale (Globicephala melas). Further, the brain transcriptomes of cattle, minke whale (Balaenoptera acutorostrata) and bowhead whale (Balaena mysticetus), which were available in the databases, were included. We found a high expression of genes related to oxidative phosphorylation and the respiratory electron chain in the whale brains. In the visual cortex of whales, transcripts related to the detoxification of reactive oxygen species were more highly expressed than in the visual cortex of cattle. These findings indicate a high oxidative capacity in the whale brain that might help to maintain aerobic metabolism in periods of reduced oxygen availability during dives.
Synaptic transmission despite severe hypoxia in hippocampal slices of the deep-diving hooded seal
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Citation Excerpt :
Although arterial oxygen tension (PaO2) has not been determined during free diving in this species, other deep-diving pinnipeds (Weddell seals, Leptonychotes weddellii; northern elephant seals, Mirounga angustirostris) have been shown to display PaO2 below 20 mmHg, even during routine diving (Qvist et al., 1986; Meir et al., 2009). On this basis, it is not surprising that hooded seal brain tissue displays remarkable neuronal survival capacities in low-oxygen/low-energy situations (e.g., Folkow et al., 2008; Ramirez et al., 2011; Czech-Damal et al., 2014). Here we confirm and extend these findings by showing that hooded seal hippocampus in vitro preparations not only survived at least 3 times longer in hypoxia than previously shown for cortical slices of this species (Folkow et al., 2008), but also that they were able to maintain synaptic transmission for this duration (Fig. 3).
Brain neurons of the deep-diving hooded seal (Cystophora cristata) are known to be inherently hypoxia tolerant. Here, we have used in vitro field potential recordings in hippocampal slices to compare effects of severe hypoxia on synaptic transmission in hooded seals vs. non-diving mammals. Synaptic responses of mice (Mus musculus) to hypoxia were in accordance with previously published data. Hippocampal slices of reindeer (Rangifer tarandus), an alternative large-mammal non-diving model, behaved in a similar way as mouse slices, in that synaptic activity disappeared rapidly without recovery after >20 min in hypoxia. The synaptic activity of hooded seal slices decreased in hypoxia, but unlike mice and reindeer, it remained at >30% of the normoxic amplitude throughout 3 h of severe hypoxia. Also, upon reoxygenation, the signal recovered to ∼50% of the pre-challenge (normoxic) amplitude. The AMPA-type glutamate receptor antagonist CNQX eliminated this signal, showing that it was not an artifact. Paired pulse facilitation (PPF), typically associated with increased presynaptic calcium (Ca²⁺) levels, was significantly reduced in the seal slices. We propose that the build-up of Ca²⁺ concentration is limited in seal presynaptic terminals, possibly due to a high Ca²⁺ buffering capacity, which could explain both the attenuated PPF and the remarkable neural hypoxia tolerance of this species. Although we found no significant hypoxia-induced upregulation of mRNA for the Ca²⁺ binding proteins calbindin d28k or parvalbumin in hooded seal hippocampal slices, a recent study reports very high transcript levels of the Ca²⁺ binding protein S100B in this species, which is in support of the hypothesis.
K<inf>ATP</inf>-channels play a minor role in the protective hypoxic shut-down of cerebellar activity in eider ducks (Somateria mollissima)
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Eider duck (Somateria mollissima) cerebellar neurons are highly tolerant toward hypoxia in vitro, which in part is due to a hypoxia-induced depression of their spontaneous activity. We have studied whether this response involves ATP-sensitive potassium (K_ATP) channels, which are known to be involved in the hypoxic/ischemic defense of mammalian neural and muscular tissues, by causing hyperpolarization and reduced ATP demand. Extracellular recordings in the Purkinje layer of isolated normoxic eider duck cerebellar slices showed that their spontaneous neuronal activity decreased significantly compared to in control slices when the K_ATP channel opener diazoxide (600 μM) was added (F_1,70 = 92.781, p < 0.001). Adding the K_ATP channel blocker tolbutamide (400 μM) 5 min prior to diazoxide completely abolished its effect (F_1,55 = 39.639, p < 0.001), strongly suggesting that these drugs have a similar mode of action in this avian species as in mammals. The spontaneous activity of slices treated with tolbutamide in combined hypoxia/chemical anoxia (95% N₂–5% CO₂ and 2 mM NaCN) was not significantly different from that of control slices (F_1,203 = 0.071, p = 0.791). Recovery from hypoxia/anoxia was, however, slightly but significantly weaker in tolbutamide-treated slices than in control slices (F_1,137 = 15.539, p < 0.001). We conclude that K_ATP channels are present in eider duck cerebellar neurons and are activated in hypoxia/anoxia, but that they do not play a key role in the protective shut-down response to hypoxia/anoxia.
The role of glycogen, glucose and lactate in neuronal activity during hypoxia in the hooded seal (Cystophora cristata) brain
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Also, previous studies using other techniques have demonstrated an enhanced enzymatic capacity for anaerobic glycolysis in diving species (Messelt and Blix, 1976; Shoubridge et al., 1976), including seals (Murphy et al., 1980). In agreement with previous results (Ramirez et al., 2007, 2011; Folkow et al., 2008), this study shows that the brain neurons of the hooded seal display a high hypoxia tolerance. Here we show that the neurons of the hooded seal are also more tolerant toward lactate and changes in exogenous substrate availability.
The brains of diving mammals are repeatedly exposed to hypoxic conditions during diving. Brain neurons of the hooded seal (Cystophora cristata) have been shown to be more hypoxia tolerant than those of mice, but the underlying mechanisms are not clear. Here we investigated the roles of different metabolic substrates for maintenance of neuronal activity and integrity, by comparing the in vitro spontaneous neuronal activity of brain slices from layer V of the visual cortex of hooded seals with those in mice (Mus musculus). Studies were conducted by manipulating the composition of the artificial cerebrospinal fluid (aCSF), containing either 10 mM glucose, or 20 mM lactate, or no external carbohydrate supply (aglycemia). Normoxic, hypoxic and ischemic conditions were applied. The lack of glucose or the application of lactate in the aCSF containing no glucose had little effect on the neuronal activity of seal neurons in either normoxia or hypoxia, while neurons from mice survived in hypoxia only few minutes regardless of the composition of the aCSF. We propose that seal neurons have higher intrinsic energy stores. Indeed, we found about three times higher glycogen stores in the seal brain (∼4.1 ng per μg total protein in the seal cerebrum) than in the mouse brain. Notably, in aCSF containing no glucose, seal neurons can tolerate 20 mM lactate while in mouse neuronal activity vanished after few minutes even in normoxia. This can be considered as an adaptation to long dives, during which lactate accumulates in the blood.
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Resistance to tissue hypoxia is a robust fundamental adaptation to low oxygen supply, and represents a novel neuroscience problem with significance to mammalian physiology as well as human health. With the underlying mechanisms strongly conserved in evolution, the ability to resist tissue hypoxia in natural systems has recently emerged as an interesting model in mammalian physiology research to understand mechanisms that can be manipulated for the clinical management of stroke. The extraordinary ability to resist tissue hypoxia by the naked mole rat (NMR) indicates the presence of a unique mechanism that underlies the remarkable healthy life span and exceptional hypoxia resistance. This opens an interesting line of research into understanding the mechanisms employed by the naked mole rat (Heterocephalus glaber) to protect the brain during hypoxia. In a series of studies, we first examined the presence of neuroprotection in the brain cells of naked mole rats (NMRs) subjected to hypoxic insults, and then characterized the expression of such neuroprotection in a wide range of time intervals. We used oxygen nutrient deprivation (OND), an in vitro model of resistance to tissue hypoxia to determine whether there is evidence of neuronal survival in the hippocampal (CA1) slices of NMRs that are subjected to chronic hypoxia. Hippocampus neurons of NMRs that were kept in hypoxic condition consistently tolerated OND right from the onset time of 5 h. This tolerance was maintained for 24 h. This finding indicates that there is evidence of resistance to tissue hypoxia by CA1 neurons of NMRs. We further examined the effect of hypoxia on metabolic rate in the NMR. Repeated measurement of metabolic rates during exposure of naked mole rats to hypoxia over a constant ambient temperature indicates that hypoxia significantly decreased metabolic rates in the NMR, suggesting that the observed decline in metabolic rate during hypoxia may contribute to the adaptive mechanism used by the NMR to resist tissue hypoxia. This work is aimed to contribute to the understanding of mechanisms of resistance to tissue hypoxia in the NMR as an important life-sustaining process, which can be translated into therapeutic interventions during stroke.

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