Circadian coupling of mitochondria in a deep-diving mammal.

Regulation of mitochondrial oxidative phosphorylation is essential to match energy supply to changing cellular energy demands, and to cope with periods of hypoxia. Recent work implicates the circadian molecular clock in control of mitochondrial function and hypoxia sensing. Because diving mammals experience intermittent episodes of severe hypoxia, with diel patterning in dive depth and duration, it is interesting to consider circadian-mitochondrial interaction in this group. Here, we demonstrate that the hooded seal (Cystophora cristata), a deep-diving Arctic pinniped, shows strong daily patterning of diving behaviour in the wild. Cultures of hooded seal skin fibroblasts exhibit robust circadian oscillation of the core clock genes per2 and arntl. In liver tissue collected from captive hooded seals, expression of arntl was some 4-fold higher in the middle of the night than in the middle of the day. To explore the clock-mitochondria relationship, we measured the mitochondrial oxygen consumption in synchronized hooded seal skin fibroblasts and found a circadian variation in mitochondrial activity, with higher coupling efficiency of complex I coinciding with the trough of arntl expression. These results open the way for further studies of circadian-hypoxia interactions in pinnipeds during diving.

Structure-function relationships in the nasal cavity of Arctic and subtropical seals.

The heating and moistening of inhaled air, and the cooling and moisture removal from exhaled air, are crucial for the survival of animals under severe environmental conditions. Arctic mammals have evolved specific adaptive mechanisms to retain warmth and water and restrict heat loss during breathing. Here, the role of the porous turbinates of the nasal cavities of Arctic and subtropical seals is studied with this in mind. Mass and energy balance equations are used to compute the time-dependent temperature and water vapor profiles along the nasal passage. A quasi-1D model based on computed tomography images of seal nasal cavities is used in numerical simulations. Measured cross-sectional areas of the air channel and the perimeters of the computed tomography slices along the nasal cavities of the two seal species are used. The model includes coupled heat and vapor transfer at the air-mucus interface and heat transfer at the interfaces between the tissues and blood vessels. The model, which assumes constant blood flow to the nose, can be used to predict the temperature of the exhaled air as a function of ambient temperature. The energy dissipation (entropy production) in the nasal passages was used to measure the relative importance of structural parameters for heat and water recovery. We found that an increase in perimeter led to significant decreases in the total energy dissipation. This is explained by improved conditions for heat and water transfer with a larger complexity of turbinates. Owing to differences in their nasal cavity morphology, the Arctic seal is expected to be advantaged in these respects relative to the subtropical seal.

The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

Thermal modeling of the respiratory turbinates in arctic and subtropical seals.

Mammals possess complex structures in their nasal cavities known as respiratory turbinate bones, which help the animal to conserve body heat and water during respiratory gas exchange. We considered the function of the maxilloturbinates of two species of seals, one arctic (Erignathus barbatus), one subtropical (Monachus monachus). By means of a thermo-hydrodynamic model that describes the heat and water exchange in the turbinate region we are able to reproduce the measured values of expired air temperatures in grey seals (Halichoerus grypus), a species for which experimental data are available. At the lowest environmental temperatures, however, this is only possible in the arctic seal, and only if we allow for the possibility of ice forming on the outermost turbinate region. At the same time the model predicts that for the arctic seals, the inhaled air is brought to deep body temperature and humidity conditions in passing the maxilloturbinates. The modeling shows that heat and water conservation go together in the sense that one effect implies the other, and that the conservation is most efficient and most flexible in the typical environment of both species. By controlling the blood flow through the turbinates the arctic seal is able to vary the heat and water conservation substantially at its average habitat temperatures, but not at temperatures around -40 °C. The subtropical species has simpler maxilloturbinates, and our model predicts that it is unable to bring inhaled air to deep body conditions, even in its natural environment, without some congestion of the vascular mucosa covering the maxilloturbinates. Physiological control of both blood flow rate and mucosal congestion is expected to have profound effects on the heat exchange function of the maxilloturbinates in seals.

Tissue distribution of angiotensin-converting enzyme 2 (ACE2) receptor in wild animals with a focus on artiodactyls, mustelids and phocids.

Natural cases of zooanthroponotic transmission of SARS-CoV-2 to animals have been reported during the COVID-19 pandemic, including to free-ranging white-tailed deer (Odocoileus virginianus) in North America and farmed American mink (Neovison vison) on multiple continents. To understand the potential for angiotensin-converting enzyme 2 (ACE2)-mediated viral tropism we characterised the distribution of ACE2 receptors in the respiratory and intestinal tissues of a selection of wild and semi-domesticated mammals including artiodactyls (cervids, bovids, camelids, suids and hippopotamus), mustelid and phocid species using immunohistochemistry. Expression of the ACE2 receptor was detected in the bronchial or bronchiolar epithelium of several European and Asiatic deer species, Bactrian camel (Camelus bactrianus), European badger (Meles meles), stoat (Mustela erminea), hippopotamus (Hippopotamus amphibious), harbor seal (Phoca vitulina), and hooded seal (Cystophora cristata). Further receptor mapping in the nasal turbinates and trachea revealed sparse ACE2 receptor expression in the mucosal epithelial cells and occasional occurrence in the submucosal glandular epithelium of Western roe deer (Capreolus capreolus), moose (Alces alces alces), and alpaca (Vicunga pacos). Only the European badger and stoat expressed high levels of ACE2 receptor in the nasal mucosal epithelium, which could suggest high susceptibility to ACE2-mediated respiratory infection. Expression of ACE2 receptor in the intestinal cells was ubiquitous across multiple taxa examined. Our results demonstrate the potential for ACE2-mediated viral infection in a selection of wild mammals and highlight the intra-taxon variability of ACE2 receptor expression, which might influence host susceptibility and infection.

Pharmacokinetics of a long-acting subcutaneous eprinomectin injection in semi-domesticated reindeer (Rangifer tarandus tarandus) - A pilot study.

Reindeer (Rangifer tarandus tarandus) are exposed to the pathogenic parasitic nematode Elaphostrongylus rangiferi during grazing. The severity of disease is dose-dependent. Prophylactic anthelmintic treatment is needed to improve animal health and reindeer herding sustainability. Herds are traditionally only gathered once during the summer, requiring a drug with a persistent effect. In this study we investigated the suitability of long-acting eprinomectin, given as a single subcutaneous injection at 1 mg/kg bodyweight in adult reindeer and calves. Plasma and faeces concentrations were determined using ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS). Plasma concentrations remained above the presumed effect level of 2 ng/mL for 80 days, demonstrating the drug's potential. Pharmacokinetic parameters were compared to other species using allometric scaling. Calves and adults had slightly different profiles. No viable faecal nematode eggs were detected during treatment. Eprinomectin was measurable in the reindeer faeces up to 100 days, which is of environmental concern.

Transcriptomes Suggest That Pinniped and Cetacean Brains Have a High Capacity for Aerobic Metabolism While Reducing Energy-Intensive Processes Such as Synaptic Transmission.

The mammalian brain is characterized by high energy expenditure and small energy reserves, making it dependent on continuous vascular oxygen and nutritional supply. The brain is therefore extremely vulnerable to hypoxia. While neurons of most terrestrial mammals suffer from irreversible damage after only short periods of hypoxia, neurons of the deep-diving hooded seal (Cystophora cristata) show a remarkable hypoxia-tolerance. To identify the molecular mechanisms underlying the intrinsic hypoxia-tolerance, we excised neurons from the visual cortices of hooded seals and mice (Mus musculus) by laser capture microdissection. A comparison of the neuronal transcriptomes suggests that, compared to mice, hooded seal neurons are endowed with an enhanced aerobic metabolic capacity, a reduced synaptic transmission and an elevated antioxidant defense. Publicly available whole-tissue brain transcriptomes of the bowhead whale (Balaena mysticetus), long-finned pilot whale (Globicephala melas), minke whale (Balaenoptera acutorostrata) and killer whale (Orcinus orca), supplemented with 2 newly sequenced long-finned pilot whales, suggest that, compared to cattle (Bos taurus), the cetacean brain also displays elevated aerobic capacity and reduced synaptic transmission. We conclude that the brain energy balance of diving mammals is preserved during diving, due to reduced synaptic transmission that limits energy expenditure, while the elevated aerobic capacity allows efficient use of oxygen to restore energy balance during surfacing between dives.

Environmental drivers of population-level variation in the migratory and diving ontogeny of an Arctic top predator.

The development of migratory strategies that enable juveniles to survive to sexual maturity is critical for species that exploit seasonal niches. For animals that forage via breath-hold diving, this requires a combination of both physiological and foraging skill development. Here, we assess how migratory and dive behaviour develop over the first year of life for a migratory Arctic top predator, the harp seal Pagophilus groenlandicus, tracked using animal-borne satellite relay data loggers. We reveal similarities in migratory movements and differences in diving behaviour between 38 juveniles tracked from the Greenland Sea and Northwest Atlantic breeding populations. In both regions, periods of resident and transitory behaviour during migration were associated with proxies for food availability: sea ice concentration and bathymetric depth. However, while ontogenetic development of dive behaviour was similar for both populations of juveniles over the first 25 days, after this time Greenland Sea animals performed shorter and shallower dives and were more closely associated with sea ice than Northwest Atlantic animals. Together, these results highlight the role of both intrinsic and extrinsic factors in shaping early life behaviour. Variation in the environmental conditions experienced during early life may shape how different populations respond to the rapid changes occurring in the Arctic ocean ecosystem.

Elevated antioxidant defence in the brain of deep-diving pinnipeds.

While foraging, marine mammals undertake repetitive diving bouts. When the animal surfaces, reperfusion makes oxygen readily available for the electron transport chain, which leads to increased production of reactive oxygen species and risk of oxidative damage. In blood and several tissues, such as heart, lung, muscle and kidney, marine mammals generally exhibit an elevated antioxidant defence. However, the brain, whose functional integrity is critical to survival, has received little attention. We previously observed an enhanced expression of several antioxidant genes in cortical neurons of hooded seals (Cystophora cristata). Here, we studied antioxidant gene expression and enzymatic activity in the visual cortex, cerebellum and hippocampus of harp seals (Pagophilus groenlandicus) and hooded seals. Moreover, we tested several genes for positive selection. We found that antioxidants in the first line of defence, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione (GSH) were constitutively enhanced in the seal brain compared to mice (Mus musculus), whereas the glutaredoxin and thioredoxin systems were not. Possibly, the activity of the latter systems is stress-induced rather than constitutively elevated. Further, some, but not all members, of the glutathione-s-transferase (GST) family appear more highly expressed. We found no signatures of positive selection, indicating that sequence and function of the studied antioxidants are conserved in pinnipeds.

Cell Culture Experiments Reveal that High S100B and Clusterin Levels may Convey Hypoxia-tolerance to the Hooded Seal (Cystophora cristata) Brain.

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.

Energy efficiency of respiration in mature and newborn reindeer.

Reindeer (Rangifer tarandus) have evolved elaborate nasal turbinate structures that are perfused via a complex vascular network. These are subject to thermoregulatory control, shifting between heat conservation and dissipation, according to the animal's needs. The three-dimensional design of the turbinate structures is essential in the sense that they determine the efficiency with which heat and water are transferred between the structure and the respired air. The turbinates have already a relatively large surface area at birth, but the structures have yet not reached the complexity of the mature animal. The aim of this study was to elucidate the structure-function relationship of the heat exchange process. We have used morphometric and physiological data from newborn reindeer calves to construct a thermodynamic model for respiratory heat and water exchange and present novel results for the simulated respiratory energy losses of calves in the cold. While the mature reindeer effectively conserves heat and water through nasal counter-current heat exchange, the nose of the calf has not yet attained a similar efficiency. We speculate that this is probably related to structure-size limitations and more favourable climate conditions during early life. The fully developed structure-function relationship may serve as inspiration for engineering design. Simulations of different extents of mucosal vascularization suggest that the abundance and pattern of perfusion of veins in the reindeer nasal mucosa may contribute to the control of temperature profiles, such that nasal cavity tissue is sufficiently warm, but not excessively so, keeping heat dissipation within limits.

Reduced immune responsiveness contributes to winter energy conservation in an Arctic bird.

Animals in seasonal environments must prudently manage energy expenditure to survive the winter. This may be achieved through reductions in the allocation of energy for various purposes (e.g. thermoregulation, locomotion, etc.). We studied whether such trade-offs also include suppression of the innate immune response, by subjecting captive male Svalbard ptarmigan (Lagopus muta hyperborea) to bacterial lipopolysaccharide (LPS) during exposure to either mild temperature (0°C) or cold snaps (acute exposure to -20°C), in constant winter darkness when birds were in energy-conserving mode, and in constant daylight in spring. The innate immune response was mostly unaffected by temperature. However, energy expenditure was below baseline when birds were immune challenged in winter, but significantly above baseline in spring. This suggests that the energetic component of the innate immune response was reduced in winter, possibly contributing to energy conservation. Immunological parameters decreased (agglutination, lysis, bacteriostatic capacity) or did not change (haptoglobin/PIT54) after the challenge, and behavioural modifications (anorexia, mass loss) were lengthy (9 days). While we did not study the mechanisms explaining these weak, or slow, responses, it is tempting to speculate they may reflect the consequences of having evolved in an environment where pathogen transmission rate is presumably low for most of the year. This is an important consideration if climate change and increased exploitation of the Arctic would alter pathogen communities at a pace outwith counter-adaption in wildlife.

Transcriptome analysis reveals a high aerobic capacity in the whale brain.

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.

Terrestrial locomotion of the Svalbard rock ptarmigan: comparing field and laboratory treadmill studies.

Research into the terrestrial locomotion of birds is often based upon laboratory treadmill experiments. However, it is unclear how transposable these results are for birds moving in the wild. Here, using video recordings, we compared the kinematics of locomotion (stride frequency, stride length, stance phase, swing phase, duty factor) and speed range of Svalbard rock ptarmigan (Lagopus muta hyperborea) under field and laboratory treadmill conditions. Our findings indicate that the kinematics of walking and aerial running are conserved when moving on the treadmill and in the field. Differences, however, were found when grounded running under the two conditions, linked to substrate. Substrate effects were confirmed by analysing trials only moving over very hard snow. In line with laboratory treadmill energetic predictions, wild ptarmigan have a preferred speed during walking and to a lesser extent when aerial running but not when moving with a grounded running gait. The birds were also capable of a higher top speed in the field than that observed during treadmill studies. Our findings demonstrate that laboratory treadmill research provides meaningful information relevant to wild birds while highlighting the importance of understanding the substrate the animals are moving over.

Ambient temperature effects on stress-induced hyperthermia in Svalbard ptarmigan.

Stress-induced hyperthermia (SIH) is commonly observed during handling in homeotherms. However, in birds, handling in cold environments typically elicits hypothermia. It is unclear whether this indicates that SIH is differently regulated in this taxon or if it is due to size, because body temperature changes during handling in low temperatures have only been measured in small birds <0.03 kg (that are more likely to suffer high heat loss when handled). We have therefore studied thermal responses to handling stress in the intermediate-sized (0.5-1.0 kg) Svalbard ptarmigan (Lagopus muta hyperborea) in 0°C and -20°C, in winter and spring. Handling caused elevated core body temperature and peripheral vasoconstriction that reduced back skin temperature. Core temperature increased less, and back skin temperature decreased more, in -20°C than in 0°C, probably because of higher heat-loss rate at the lower temperature. Responses were qualitatively consistent between seasons, despite higher body condition/insulation in winter and dramatic seasonal changes in photoperiod, both of which could possibly affect stress responsiveness. Our study supports the notion that SIH is a general thermoregulatory reaction to acute stressors in endotherms, but also suggests that body size and thermal environment should be taken into account when evaluating this response in birds.

Seasonal variation in the thermal responses to changing environmental temperature in the world's northernmost land bird.

Arctic homeotherms counter challenges at high latitudes using a combination of seasonal adjustments in pelage/plumage, fat deposition and intricate thermoregulatory adaptations. However, there are still gaps in our understanding of their thermal responses to cold, particularly in Arctic birds. Here, we have studied the potential use of local heterothermy (i.e. tissue cooling that can contribute to significantly lower heat loss rate) in Svalbard ptarmigan (Lagopus muta hyperborea) - the world's northernmost land bird. We exposed birds kept under simulated Svalbard photoperiod to low ambient temperatures (Ta; between 0 and -30°C) during three seasons (early winter, late winter, summer), whilst recording resting metabolic rate (RMR), core temperature (Tc) and several cutaneous temperatures. Leg skin temperature varied the most, but still only by up to ∼15°C, whereas body trunk skin temperature changed <1°C when Ta decreased from 0 to -30°C. At the same time, Tc increased by 0.9°C, concomitant with increased RMR. This was probably driven by the triggering of cerebral thermosensors in response to cooling of the poorly insulated head, the skin of which was 5.4°C colder at -30°C than at 0°C. Thermal conductance in winter was higher in yearlings, probably because they were time/resource constrained from acquiring a high-quality plumage and sufficient fat reserves as a result of concomitant body growth. In conclusion, Svalbard ptarmigan do not employ extensive local heterothermy for cold protection but instead rely on efficient thermogenesis combined with excellent body insulation. Hence, cold defence in the world's northernmost land bird is not mechanistically much different from that of its lower latitude relatives.

Effects of Polar Bear and Killer Whale Derived Contaminant Cocktails on Marine Mammal Immunity.

Most controlled toxicity studies use single chemical exposures that do not represent the real world situation of complex mixtures of known and unknown natural and anthropogenic substances. In the present study, complex contaminant cocktails derived from the blubber of polar bears (PB; Ursus maritimus) and killer whales (KW; Orcinus orca) were used for in vitro concentration-response experiments with PB, cetacean and seal spp. immune cells to evaluate the effect of realistic contaminant mixtures on various immune functions. Cytotoxic effects of the PB cocktail occurred at lower concentrations than the KW cocktail (1 vs 16 µg/mL), likely due to differences in contaminant profiles in the mixtures derived from the adipose of each species. Similarly, significant reduction of lymphocyte proliferation occurred at much lower exposures in the PB cocktail (EC50: 0.94 vs 6.06 µg/mL; P < 0.01), whereas the KW cocktail caused a much faster decline in proliferation (slope: 2.9 vs 1.7; P = 0.04). Only the KW cocktail modulated natural killer (NK) cell activity and neutrophil and monocyte phagocytosis in a concentration- and species-dependent manner. No clear sensitivity differences emerged when comparing cetaceans, seals and PB. Our results showing lower effect levels for complex mixtures relative to single compounds suggest that previous risk assessments underestimate the effects of real world contaminant exposure on immunity. Our results using blubber-derived contaminant cocktails add realism to in vitro exposure experiments and confirm the immunotoxic risk marine mammals face from exposure to complex mixtures of environmental contaminants.

Transcriptome Analysis Identifies Key Metabolic Changes in the Hooded Seal (Cystophora cristata) Brain in Response to Hypoxia and Reoxygenation.

The brain of diving mammals tolerates low oxygen conditions better than the brain of most terrestrial mammals. Previously, it has been demonstrated that the neurons in brain slices of the hooded seal (Cystophora cristata) withstand hypoxia longer than those of mouse, and also tolerate reduced glucose supply and high lactate concentrations. This tolerance appears to be accompanied by a shift in the oxidative energy metabolism to the astrocytes in the seal while in terrestrial mammals the aerobic energy production mainly takes place in neurons. Here, we used RNA-Seq to compare the effect of hypoxia and reoxygenation in vitro on brain slices from the visual cortex of hooded seals. We saw no general reduction of gene expression, suggesting that the response to hypoxia and reoxygenation is an actively regulated process. The treatments caused the preferential upregulation of genes related to inflammation, as found before e.g. in stroke studies using mammalian models. Gene ontology and KEGG pathway analyses showed a downregulation of genes involved in ion transport and other neuronal processes, indicative for a neuronal shutdown in response to a shortage of O2 supply. These differences may be interpreted in terms of an energy saving strategy in the seal's brain. We specifically analyzed the regulation of genes involved in energy metabolism. Hypoxia and reoxygenation caused a similar response, with upregulation of genes involved in glucose metabolism and downregulation of the components of the pyruvate dehydrogenase complex. We also observed upregulation of the monocarboxylate transporter Mct4, suggesting increased lactate efflux. Together, these data indicate that the seal brain responds to the hypoxic challenge by a relative increase in the anaerobic energy metabolism.

Synaptic transmission despite severe hypoxia in hippocampal slices of the deep-diving hooded seal.

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 >20min 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 3h 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.